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Can any protein be phosphorylated?

Can any protein be phosphorylated?


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I am working with an Arabidopsis mutant with an F-box protein knocked out. It has been shown that F-box proteins targets must first be phosphorylated (Skowrya et al., 1997). I have heard of phosphorylation sites, but I can't find out whether every protein has them. Can any protein be phosphorylated?

  • Skowyra, D., Craig, K.L., Tyers, M., Elledge, S.J. & Harper, J.W. (1997) F-Box Proteins Are Receptors that Recruit Phosphorylated Substrates to the SCF Ubiquitin-Ligase Complex. Cell. 91 (2), 209-219.

Phosphorylation can occur on specific amino acids only, what you have called phosporylation sites. These amino acids are Ser, Tyr, Asp, Thr and His. In theory any of these amino acids may be phosphorylated, but in reality it may not actually occur for a number of reasons. Some of these are because of the change in overall charge of the protein which can influence the 3D conformation, or the amino acids are not accessible to specific kinases, etc. If you ask for the purposes of doing a Western blot, then the antibody specification sheet should indicate whether a phosphorylated form exists and there should be a reference to the literature describing this modification.


One important thing is missing in the other answers: not only phosphorylation will happen only at selected aminoacids, but it will not happen at all of those.

So, not all of the Ser/Thr/Tyr of a protein can be phosphorylated because they could be structurally unaccessible to protein kinase and because they need to be in a specific motif in order to be phosphorylated.

The Human Protein Reference Database, for instance, lists the phosphorylation motifs for many Tyr and Ser/Thr kinases.


Phosphorylation requires exposed serine, threonine, tyrosine, or histidine residues (in eukaryotes). This is because the transfer of phosphate groups to proteins is mediated by a class of proteins called kinases. Kinases can have broad or specific activity.

This review ought to have most of the answers to your questions :

http://www.cell.com/abstract/S0092-8674(06)01274-8


I like Nico's response best +1. I did find an interesting review on phosphoarginine and phospholysine - the list of possible phosphorylation sites grows. Not only is it the spatial context of the amino acid to be (or not) phosphorylated, as Nico writes and Leonardo implies, but also the temporal. Are the target protein and protein kinase co-expressed? If you're looking at specific tissues (root, flower, leaf) and the kinase in question is only produced in seedlings, maybe that potential phosphorylation won't really occur.


For one of the most comprehensive databases of protein post-translational modification (including phosphorylation, methylation, acetylation, ubiquitination, etc.), check out PhosphoSite. You can find links to sequences, diseases, motifs, publications, antibodies, mass spec experiments, structures, you name it.


PROTEIN PHOSPHORYLATION: A GLOBAL REGULATOR OF CELLULAR ACTIVITY

As early as the 19th century it was known that phosphates could be bound to proteins. Most examples of these ‘phosphoproteins’ were found in milk (caseins) and egg yolk (phosvitin) and were simply considered a biological method of providing phosphorus as a nutrient. Therefore, the existence of phosphoproteins was considered a consequence of metabolic reactions, and nothing more, for almost a century after their discovery [1].

In the 1950’s this all began to change as phosphoproteins began to emerge as key regulators of cellular life. An initiating factor of this emergence occurred in 1954, when an enzyme activity was observed that transferred a phosphate onto another protein [2] -a biological reaction called phosphorylation. The protein responsible was a liver enzyme that catalyzed the phosphorylation of casein and became known as a protein kinase (see Figure 1), the first of its kind to be discovered. A year later, the role of phosphorylation became more interesting as Fischer and Krebs [3], and Wosilait and Sutherland4, showed that an enzyme involved in glycogen metabolism was regulated by the addition or removal of a phosphate, suggesting that reversible phosphorylation could control enzyme activity. This idea was later proven to be true and has now seeped into virtually every aspect of cell biology.


Figure 1. Protein kinases and protein phosphatases.

Today, it is thought that one third of the proteins present in a typical mammalian cell are covalently bound to phosphate (i.e. they are phosphorylated at one time or another). The study of cell biology is now littered with examples of regulation by phosphorylation: increasing or decreasing the biological activity of an enzyme, helping move proteins between subcellular compartments, allowing interactions between proteins to occur, as well as labeling proteins for degradation. The variety is immense and now many human diseases have been recognized to be associated with the abnormal phosphorylation of cellular proteins.

These developments have brought the study of phosphorylation into the limelight of medical research, a fact that was recognized in 1992 when Fischer and Krebs received the Nobel Prize in medicine for their pioneering efforts. For those interested, the lectures given by Fischer and Krebs upon receiving the Noble Prize are an excellent overview of the early days of this field [5].

How can phosphorylation control enzyme activity?

Phosphorylation refers to the addition of a phosphate to one of the amino acid side chains of a protein. Remember that proteins are composed of amino acids bound together and that each amino acid contains a particular side chain, which distinguishes it from other amino acids. Phosphates are negatively charged (with each phosphate group carrying two negative charges) so that their addition to a protein will change the characteristics of the protein. This change is often a conformational one, causing the protein to change how it is structured (see Figure 2).


Figure 2. Conformation changes caused by phosphorylation.

This reaction is reversible by a process called dephosphorylation. The protein switches back to its original conformation when the phosphorus is removed (see Figure 2). If these two conformations provide the protein with different activities (i.e. being enzymatically active in one conformation but not the other), phosphorylation of the protein will act as a molecular switch, turning the activity on or off.

The transfer of phosphates onto proteins is catalyzed by a variety of enzymes in the cell. Although the variety is large, all of these enzymes share certain characteristics and fall into one class of proteins, called protein kinases [6]. Their similarities stem from the group’s ability to take a phosphate off the chemical energy-carrying molecule ATP and place it onto an amino acid side chain of a protein (see Figure 3). The hydroxyl groups (-OH) of serine, threonine, tyrosine or histidine amino acid side chains are the most common target. A second class of enzymes is responsible for the reverse reaction, in which phosphates are removed from a protein. These are termed protein phosphatases.


Figure 3. Addition of a phosphate to an amino acid.

The use of the phosphorylation/dephosphorylation of a protein as a control mechanism has many advantages:

  • It is rapid, taking as little as a few seconds.
  • It does not require new proteins to be made or degraded.
  • It is easily reversible.

The extensive use of this control mechanism is apparent by the large number of known kinases and phosphatases [6]. Even in a simple organism like yeast, approximately 3 percent of its proteins are kinases or phosphatases. Some of these enzymes are extremely specific, potentially phosphorylating or dephosphorylating only a few target proteins, while others are able to act broadly on many proteins. The examples of known targets of phosphorylation include most protein components of the cell, including enzymes, structural proteins, cell receptors, ion channels and signaling molecules. If a protein is controlled by its phosphorylation state, its activity at any one time will be directly dependent on the activity of the kinases and phosphatases that act on it. It is quite common for a phosphate group to be added or removed from a protein continually, a cycle that allows a protein to switch rapidly from one state to another.

External Signals can activate protein kinases and phosphatases

The reception of a signal on the surface of a cell often results in the activation of kinases and phosphatases [6]. Once activated, cellular phosphorylation patterns will begin to change, with various proteins being phosphorylated or dephosphorylated. The final result will be various changes in cellular behavior.

Many of the proteins that are phosphorylated upon reception of a signal are protein kinases as well. This organization of kinases produces a phosphorylation cascade [6] (see Figure 4), in which one protein kinase is activated by phosphorylation upon reception of a signal, this kinase then phosphorylates the next kinase in the cascade, and so on until the signal is transmitted through the cell. In such a system, the kinase cascade can start with the receptor itself (which is often a kinase) or a free-floating cytoplasmic kinase. Upon reception of a signal, these phosphorylation cascades continue to function until protein phosphatases are activated and shut off their transmission.


Figure 4. Phosphorylation Cascades

In animal cells, these cascades are mediated by two types of kinases: serine/threonine kinases [6] (which phosphorylate serine and threonine amino acid side chains) and tyrosine kinases [6] (which phosphorylate tyrosine amino acid side chains).

Phosphorylation in response to a signal produces a second outcome apart from the activation of kinases and phosphatases, which involves the production of binding sites for proteins to interact [6]. This process is different from the activation of a protein by phosphorylation (in which the addition of a phosphate causes differences in enzyme activity), since it does not necessarily change the inherent activity of the molecule that has been phosphorylated. Instead, it creates a phosphorylated amino acid on the molecule that another protein can bind to (see Figure 4). Upon reception of a signal, some membrane-bound receptors will become tyrosine phosphorylated. Free-floating proteins then bind to these phosphotyrosine sites and are thus concentrated near the receptor. This concentration often leads to the activation of additional proteins by bringing together molecules that normally would not be in close proximity.

Through the use of phosphorylation cycles and cascades, the cell is able to regulate a diverse set of processes, including cellular movement, reproduction and metabolism. It is the simplicity, reversibility and flexibility of phosphorylation that explains why it has been adopted as the most general control mechanism of the cell.

Addition Reading And Texts Consulted

1. Pawson T. 1994. Introduction: Protein Kinases. FASEB J. 8:1112-1113.

2. Hardie DG, ed. 1999. Protein Phosphorylation: A Practical Approach. Oxford/New York: Oxford University Press. 431p.

3. Hardie DG, Hanks S, eds. 1995. The Protein Kinase Factsbook. London/San Diego: Academic Press. Vol. 1-2.

1. Marks F, ed. 1996. Protein Phosphorylation. Weinheim: VCH.

2. Burnett G, Kennedy EP. 1954. J. Biol. Chem. 211: 969-980.

3. Fischer EH, Krebs EG. 1955. J. Biol. Chem. 216: 121-132.

4. Sutherland EW, Wosilait WD. 1955. Nature 175: 169-170.

6. Sefton BM, Hunter T. 1998. Protein Phosphorylation. San Diego: Academic Press.

(Art by Jane Wang – note that high res versions of image files available here)


Serine/Threonine Protein Phosphatases

Introduction

Reversible protein phosphorylation is an ancient, universal and crucial means to dynamically and coordinately regulate cellular homeostasis and the cellular response to external signals. The biochemical basis for the regulatory capacity of protein (de)phosphorylations is the addition or removal of a negatively charged phosphate to a protein, which locally and globally may change the structure of the modified protein, and like that, affects the biological activity of that protein. The phosphorylation balance is crucial and is determined by the activities of protein kinases and protein phosphatases, which catalyze protein phosphorylations and dephosphorylations, respectively, and act as signal transducers in their own right ( Fig. 1 ). Dysregulation of this balance inevitably leads to disease hence, both protein kinases and protein phosphatases harbor significant therapeutic potential in cancer, diabetes, cardiovascular disease, immune disease, neurodegenerative diseases and many other human pathologies. Proteomics studies revealed that in normal cells protein (de)phosphorylations occur in 86.4% of cases on serine, in 11.8% of cases on threonine, and in only 1.8% of cases on tyrosine residues ( Olsen et al., 2006 ). This also reflects the historical classification of protein phosphatases into Protein Serine/Threonine Phosphatases (PSTPs or PSPs) and Protein Tyrosine Phosphatases (PTPs). These two main phosphatase classes became each further subdivided into different superfamilies. For the PSPs, these are the phosphoprotein phosphatases (PPP), the metal-dependent protein phosphatases (PPM), and the Asp-based FCP (TFIIF-associating component of RNA polymerase II C-terminal domain) and SCP (small C-terminal domain) phosphatases ( Shi, 2009 ) ( Fig. 1 ).

Fig. 1 . Protein phosphorylation and Ser/Thr protein phosphatases. Reversible protein phosphorylation occurs on a serine, threonine or tyrosine residue and requires the activity of protein kinases and protein phosphatases, which catalyze protein phosphorylation and dephosphorylation, respectively. In a normal cell, the bulk of protein phosphorylations (98.2%) occur on serine and threonine residues, while only 1.8% of phosphorylations occur on tyrosine residues. The fully sequenced human genome is thought to contain 38 Ser/Thr protein phosphatases (PSPs), which are divided into three superfamilies, denoted PPP, PPM and SCP/FCP.


Nociceptin Opioid

2.2 Identification of prepronociceptin gene associating cAMP-dependently with phosphorylated CREB

To identify novel genes that are regulated by CREB phosphorylated at Ser133 in Sertoli cells, we performed chromatin immunoprecipitation (ChIP) from Sertoli B cells. After cells were stimulated for 10 min with db-cAMP, extracts were prepared and processed for ChIP with the same antibody to phosphorylated CREB. We screened by PCR several genes, whose proximal promoter regions associate with phosphorylated CREB, and investigated murine prepronociceptin gene ( Zaveri, Waleh, & Toll, 2006 ). The proximal promoter of murine prepronociceptin gene has one functional CRE site in a different location from the human promoter ( Zaveri, Green, Polgar, Huynh, & Toll, 2002 Zaveri et al., 2006 ). The DNA fragment from the putative transcription start site to the ATG translation start codon (252 bp) was detected only in cells treated with db-cAMP but not in untreated cells. None could be detected from immunoprecipitates with an unrelated antibody. Nucleotide sequencing of the detected DNA fragment confirmed the presence of a consensus CRE sequence (CGTCA) at 30 bp upstream of the ATG translation start codon in the proximal promoter of murine prepronociceptin gene as reported ( Zaveri et al., 2006 ). These results indicated that phosphorylated CREB associates with the proximal promoter region of prepronociceptin gene in Sertoli B cells ( Fig. 1 ). This gene encodes a precursor protein of prepronociceptin, from which the mature nociceptin peptide consisting of 17 amino acid residues is produced. Nociceptin, also known as orphanin FQ, is a neuropeptide belonging to the opioid peptide family and shares the identical amino acid sequence between mice and other species.


Phosphorylation: The Master Switch of the Cell

It is no coincidence that one cellular process is mentioned time and time again in discussions of cell-signaling pathways in cancer. Since its discovery, phosphorylation has come to be recognized as a global regulator of cellular activity, and abnormal phosphorylation is implicated in a host of human diseases.

In this report, we probe a little deeper to understand what exactly protein phosphorylation does why it is such a vital, ubiquitous process and how it continues to further our understanding of diseases such as cancer.

A Minor Modification With a Major Role

Once a gene is expressed and translated into a functional cellular protein, the cell is able to control the protein’s fate through the use of posttranslational modifications (PTMs). Phosphorylation is the most important and most thoroughly researched form of PTM.

Phosphorylation of a protein involves the enzymatically mediated addition of a phosphate group (PO4) to its amino acid side chains. Phosphorylated proteins were observed as far back as the early 1900s, but it was not until the 1950s that the pioneering, and ultimately Nobel Prizewinning, discoveries of Edmond H. Fischer and Edwin G. Krebs determined that phosphorylation was a reversible, enzymatically mediated process, capable of modifying the function of a protein.

Today, it is believed that as many as one-third of all proteins in the cell are phosphorylated at one time or another, and half of these proteins likely harbor more than 1 phosphorylation site, with different sites often eliciting quite different cellular responses.

Phosphorylation and the reverse reaction, dephosphorylation, occur thanks to the actions of 2 key enzymes. Protein kinases phosphorylate proteins by transferring a phosphate group from adenosine triphosphate (ATP) to their target protein. This process is balanced by the action of protein phosphatases, which can subsequently remove the phosphate group. The amount of phosphate that is associated with a protein is therefore precisely determined by the relative activities of the associated kinase and phosphatase. As much as 2% to 5% of the human genome is thought to encode protein kinases and phosphatases.

The most common amino acids to be phosphorylated on eukaryotic proteins (proteins found in all organisms except bacteria) are serine, threonine, and tyrosine.

Functions of Phosphorylation Are Varied

At the level of a single protein, the binding of a negatively charged phosphate group can lead to changes in the structure of a protein, which alter the way that it functions. If the targeted protein is an enzyme, phosphorylation and dephosphorylation can impact its enzymatic activity, essentially acting like a switch, turning it on and off in a regulated manner.

Another outcome of structural changes to the phosphorylated protein is the facilitation of binding to a partner protein. In this way, phosphorylation can regulate protein-protein interactions. The phosphorylation of a protein can also target it for degradation and removal from the cell by the ubiquitin-proteasome system.

Protein phosphorylation also has a vital role in intracellular signal transduction. Many of the proteins that make up a signaling pathway are kinases, from the tyrosine kinase receptors at the cell surface to downstream effector proteins, many of which are serine/threonine kinases.

In a nutshell, ligand binding at the cell surface establishes a phosphorylation cascade, with the phosphorylation and activation of 1 protein stimulating the phosphorylation of another, subsequently amplifying a signal and transmitting it through the cell. The signal continues to propagate until it is switched off by the action of a phosphatase.

In addition to proteins, other kinds of molecules can also be phosphorylated. In particular, the phosphorylation of phosphoinositide lipids, such as phosphatidylinositol-4,5-bisphosphate (PIP2), at various positions on their inositol ring, also plays a key role in signal transduction.

Defining the Role of Phosphorylation in Cancer

Phosphorylation plays a vital role in regulating many intracellular processes such as growth, proliferation, and cell division. Thus, any perturbations in the phosphorylation process are likely to drive many of the hallmarks of cancer, such as unchecked cell growth and proliferation. Indeed, mutations in kinases and phosphatases are frequently implicated in a number of different cancers, and many of the genes encoding for these proteins are oncogenes or tumor suppressors.

Overexpression or mutations that lead to constitutive activation of phosphorylation machinery will inevitably disrupt its delicate balance in the cell, driving the inappropriate activation or deactivation of the cellular processes it controls.

The key function of protein kinases in signal transduction has made them an attractive target for cancer therapeutics.

Exploiting the Process in Drug Development

The key function of protein kinases in signal transduction has made them an extremely attractive target for therapeutic intervention in cancer. Protein kinases represent as much as 30% of all protein targets under investigation by pharmaceutical companies. Targeting tyrosine kinases in particular is a popular approach, and ground-breaking advances have been made in recent decades with the introduction of this class of agents.

They include drugs such as trastuzumab (Herceptin, Genentech), a monoclonal antibody designed to block the function of the HER2 receptor tyrosine kinase, which revolutionized the treatment of breast cancer, and erlotinib (Tarceva, Genentech), a receptor tyrosine kinase inhibitor, that targets the epidermal growth factor receptor (EGFR) and is approved by the FDA for treatment of patients with non-small cell lung cancer.

More recently, the serine/threonine kinases have also emerged as strong candidates and around one-third of all kinase inhibitors currently in development target serine/threonine kinases. Most advanced among this class of drugs are those targeting the mammalian target of rapamycin (mTOR): everolimus (Afinitor, Novartis) and temsirolimus (Torisel, Pfizer), both approved for the treatment of renal cell carcinoma.

A number of inhibitors targeting AKT are also in the pipeline, including perifosine (Keryx Biopharmaceuticals, Inc), and there is a substantial amount of interest in drugs targeting MEK, a critical kinase at the junction of several biological pathways that regulate cell proliferation, survival, migration, and differentiation, including AZD6244 (selumetinib, AstraZeneca).

Serine/threonine kinases involved in cell cycle regulation are also heavily investigated, such as the aurora kinases and the cyclin-dependent kinases. Among them are AZD1152 (AstraZeneca) and HMR-1275 (alvocidib sanofi -aventis), which are in various stages of clinical development.

Probing Potential as Biomarker

Given the pivotal roles of phosphorylation in the cellular environment, and the fact that the overwhelming majority of phosphorylation sites remain uncharacterized, there is a constant effort by researchers to better understand the role of phosphorylation and to develop novel, highly sensitive, and sophisticated phosphorylation identification techniques.

The gold-standard method for measuring protein phosphorylation levels is an in vitro kinase assay, in which radioactive ATP is used. When a phosphorylation event occurs, the radioactive inorganic phosphate (32P) in ATP will be transferred to the phosphorylated protein thus, following the radioactivity allows researchers to establish when proteins are phosphorylated. The development of specific antibodies against phosphorylated forms of proteins led to the use of immunoassays to examine phosphorylation patterns of individual proteins.

More recently, researchers have begun to make use of increasingly sophisticated mass spectrometry and nuclear magnetic resonance imaging methods to try to decipher global patterns of phosphorylation within the cell in response to certain stimuli or therapeutic agents. The potential for using the phosphorylation status of key signaling proteins as a diagnostic biomarker, predictor of response, or prognostic indicator is an area of intense interest, reflected in a number of presentations at this year’s American Society of Clinical Oncology (ASCO) meeting.

Studies are examining the utility of phosphorylated forms of epidermal growth factor receptor and HER2 as both biomarkers of response to tyrosine kinase inhibitors and as prognostic indictors. For example, 1 study found that a high level of expression of phosphorylated HER2 in patients with HER2- positive breast cancer is associated with lower 5-year disease-free survival.

Furthermore, researchers are beginning to exploit binding domains within signaling proteins that specifically recognize and bind to other phosphorylated proteins. Using the Src homology 2 (SH2) -binding domain, which binds to phosphorylated tyrosines, researchers have been able to analyze the global state of tyrosine phosphorylation in different human cancer cell lines to see how it differs from normal cells.

Jane de Lartigue, PhD, is a freelance medical writer and editor based in the United Kingdom.


Exogenous and Endogenous Protein Metabolism | Biology

By the term endogenous protein metabolism is meant the disintegration of those proteins which already exist as components of living cells (tissue proteins). The term exogenous protein metabolism implies the breakdown of food proteins which do not exist as parts of the cell protoplasm.

The classical view of protein metabolism, originally proposed by Folin, is now challenged. Folin’s view was the metabolic patterns of exogenous (dietary) and endogenous (body) protein metabolism differ, i.e., the end products of endogenous protein metabolism are uric acid, creatine and neutral sulphur whereas urea is the end product of exogenous protein metabolism.

However isotopic experiment reveals that the body proteins are in a constant state of turnover and the body proteins are continually broken down and replaced by new proteins synthesized from dietary amino acids. The replacement of protein is rapid in plasma, liver, kidneys and intestinal tract and slow in haemoglobin, muscle and skin.

Dynamic State of Amino Nitrogen and Proteins of the Body:

It has been established by the work of Schoenheimer and others that the amino nitrogen of amino acids (except lysine) is distributed to other amino acids of different tissues and reversibly it is withdrawn from those amino acids to the amino acids which contained amino nitrogen by the process of deamination and reamination.

Protein Storage (Labile Protein):

Nitrogen excreted for the first few days after protein starvation was greater and then becomes more or less con­stant. The disorganized protein present in liver, thymus, prostate, seminal vesicle, alimentary tract, pancreas, spleen and kidneys, etc., are drawn upon to meet the need of the body and these proteins are called labile pro­teins. These are utilized for the synthesis of other proteins and may be oxidized to gain energy when required.

End Products of Protein Metabolism:

The nitrogen released from amino acid (protein) catabolism is excreted from the body in different form which varies in different species of animals. The considerable amount of nitrogen excreted through the urine of men, mammals and amphibians, etc., is urea. The birds and reptiles excrete nitrogen as uric acid mainly. The man also excretes nitrogen of purine bases as uric acid. Creatine excretion is exclusively from tissue protein breakdown. Neutral sulphur excretion does not indicate the nature of dietary or tissue protein breakdown.

Due to dynamic state of amino nitrogen of amino acid and differential pattern of metabolism of labile protein, the excretion of different nitrogenous metabolic end product does not always give real metabolic picture of protein metabolism. But some clue may be obtained if the standardized experiments were done e.g., increased creatine excretion, may indicate increased tissue protein, i.e., specially muscle protein catabolism going on. Increased uric acid excretion may be due to increased purine catabolism. Increased urea excretion may indicate dietary protein catabolism.

Brief Life History of the End Products of Exogenous Protein Metabolism:

From Folin’s experiment, although challenged, it appears that the end products of exogenous protein metab­olism are urea, ammonia, inorganic sulphate and 50% of the total uric acid excreted.

The brief life history of these products is mentioned below:

(a) From deamination of amino acids (mainly from exogenous sources),

(b) From salts like ammonium carbonate, lactate, etc., taken in diet or as drug, and

(c) From the amino acid arginine.

It breaks down into urea and ornithine. The end product of metabolism of these bodies is urea.

20-40 mgm (average 30 mgm) of urea present per 100 ml of blood. Almost equally distributed in plasma and corpuscles.

Functions Served by Urea Formation:

Urea formation helps to maintain the reaction of blood constant. Because, in it, one acid (carbonic acid) and two molecules of ammonia remain neutralized.

An adult taking a normal mixed diet excretes urea through urine an average of 30 gm daily (2% if total urine volume be 1,500 ml). 80% of urinary nitrogen is excreted in the form of urea.

(a) From deamination of amino acids, both exogenous and endogenous. Although deamination takes place chiefly in the liver, recent observations indicate that ammonia is also formed in the kidneys. Ammonia formed in the liver is converted into various substances. Kidneys can deaminate amino acids normally. The amount increases during acidosis and falls in alkalosis, and

(b) Certain ammonium salts taken in food or as drug, e.g., ammonium chloride.

Fate and Functions of Ammonia:

(a) Form Ammonium Salts. The purpose served is to keep the blood reaction constant,

(b) Ammonia may be utilized for the synthesis of amino acid, uric acid, nucleoproteins and other nitrogenous compounds.

With a mixed diet in an adult man the total daily output is about 0.7 gm. Ammonia nitrogen constitutes about 2-4% of total urinary nitrogen.

Relation with Blood Reaction:

In acidosis more ammonium salts will be formed. Reverse changes will take place in alkalosis.

Significance of Variations:

Although ammonia is an end product of exogenous protein metabolism, yet its amount in the urine is determined by the relative proportion of acids and bases in the body. In conditions of acidosis it rises, in alkalosis it falls. Ammonia coefficient is a reliable guide to the condition of acidosis or alkalosis of the body.

They are greatly produced from dietary proteins in the body. Consequently, they may be taken as the end products of exogenous protein metabolism. (The ethereal sulphates have a different life history altogether and has been discussed under ‘Sulphur Metabolism’).

It is an index of both endogenous and exogenous protein metabolism.

Brief Life History of the End Products of Endogenous Protein Metabolism:

From dietetic experiments in dogs it has been found that creatinine and neutral sulphur remain absolutely unchanged. So they are solely of endogenous origin. In this connection, the compound creatine, although not shown in this experiment, but when present in urine, should be regarded as derived from endogenous protein metabolism, because it is the precursor of creatinine.

Thus these compounds are wholly endogenous. Half of uric acid is of endogenous origin and the other half is of exogenous origin. The life history of neutral sulphur has been discussed under Sulphur Metabolism and of uric acid under Uric Acid Metabolism.

A brief summary of the life history of creatine and creatinine is given below:

Methyl guanidoacetic acid.

Total Amount in the Body:

90-120 gm in adult, 98% of it is present in the striated muscle as creatine phosphate. Skeletal muscles contain about 0.5% creatine. It is also found in heart (about half to the amount in skeletal muscles, i.e., 0.25%), testes, brain and uterus, specially during pregnancy.

It is present in blood about 10 mgm per 100 ml and remain mostly in the red cells. As it is present inside the red cells, it is not filtered. Hence, it is usually not present in the urine.

Origin and Formation of Creatine:

(a) Creatine synthesis requires three amino acids, viz., arginine glycine and me­thionine (as S-adenosyl methionine),

(b) The compound guanidoacetic acid is an intermediate step in the synthesis of creatine,

(c) The methyl group of creatine is derived from methionine,

(d) The stages in creatine synthesis appear to be as follows- two organs, i.e., kidneys and liver, are also involved for the complete synthesis of creatine.

In kidneys, glycine and arginine react where amidine group (-CNHNH,) of arginine is transferred to glycine with the forma­tion of guanidoacetic acid (glycocyamine) by the enzyme transamidinase. Transamidinase enzyme is present only in kidneys and pancreas. But this enzymatic reaction mostly takes place in the kidneys.

Methylation of guanidoacetic acid takes place, in the liver, because the liver contains the enzyme guanidoacetic methyl transferase. Guanidoacetic acid is converted into creatine with the help of amino acid, methionine (active form) in presence of enzyme guani­doacetic methyl transferase and glutathione (GSH).

When methyl group of methionine is transferred to guanidoacetic acid to form creatine (methyl guanidoacetic acid), the methionine is converted into S-adenosyl homocysteine. Ac­tivation of methionine takes place by ATP when methionine is converted into S-adenosyl methionine. Recently, it has been shown that in mammals both transamidiantion and methylation reactions, involved in the synthesis of creatine, take place in the pancreas.

Effects of Creatine Feeding:

If creatine is ingested in small amounts (up to 1 gm daily), none is found in the urine, but in moderate amounts (up to 5 gm daily) a little is excreted as creatine and the rest is stored. But if large amounts (20 gm) of creatine be taken, the major part (15 gm) is excreted as such, another part (4.5 gm) is retained and a small part (0.5 gm) is excreted as creatinine in the urine. This shows that creatine is not a waste product. It is useful and there is a store for it in the body.

Until this reservoir is filled up, no creatine will appear in the urine. Creatine synthesis is dependent on kidney transamidinase activity. The kidneys were thought to be the only site of the transamidinating enzyme until recently. However, recent studies have indicated that the pancreas may play a unique role in the synthesis of creatine within the mammalian body.

Interrelation with Creatinine:

These two compounds are closely interrelated. They are readily interconvertible while in solution. Creatinine is anhydride of creatine having one molecule of water less. Acid medium favours the formation of creatinine, whereas alkaline medium favours the formation of creatine. But in vivo creatinine cannot be converted into creatine, although the reverse is the rule.

i. Creatine is converted into creatine phosphate (phosphagen) which takes an essential part in the chemical changes underlying muscular contraction. Creatine, when given in moderate amounts by mouth, disap­pears completely in the body and nothing appears in the urine. This is supposed to be due to its conversion into creatine phosphate and subsequent storage in the muscles.

ii. Creatine certainly has some function in tissues other than muscles but its nature is not known.

ii. Creatine is the precursor of creatinine.

Excretion of Creatine:

Creatine is not generally present in the urine of normal adult males. But it may be excreted abnormally.

Its excretion in the urine is determined by the following factors:

Up to the age of puberty it is constantly present in the urine of both sexes. It has been suggested to be due to an increased production of creatine, induced in some unknown way, by the activity of growth impulse. It may also be due to a lower capacity of the undeveloped muscles for creatine storage. There is a third possibility in that the children possess less power to convert creatine into creatinine.

After puberty it is found intermittently in healthy a female which is not related to menstruation.

It is constantly present during pregnancy. It rises to a maximum of 1.5 gm daily after confinement and is probably derived from the involuting uterus. The sex difference of creatine excretion cannot be properly explained. That increased creatine excretion is not due to the less muscular development in females is proved by the fact that it occurs even in women who are highly trained physically. That sex has something to do here is supported by the observation that creatinuria is common in eunuchs. It may be easily induced in old people (naturally with diminished sex functions) by administration of small amount of creatine.

High protein and low carbohydrate diets increase creatine excretion. The former acts by stimulating tissue metabolism due to its high specific dynamic action. The latter acts indirectly by the absence of its sparing effects upon the breakdown of tissue protein.

v. Increased Tissue Breakdown:

In any condition that increases the breakdown of tissues, specially of striated mus­cles, as in starvation, prolonged diabetes mellitus, hyperthyroidism, fevers and other wasting diseases which increase the basal metabolic rate, the creatine excretion is increased. In certain diseases of muscles (myopathy) where muscles undergo degeneration, a large amount of creatine is excreted.

In such conditions 90% or more creatine appears in an unchanged form in the urine even when it is given by mouth in small amount. This is said to be due to a lower storage capacity of the muscle. It is also probable that in this disease (i.e., myopathy) the reversible enzyme reaction, by which the broken creatine phosphate becomes re-synthesised in the muscle, is absent.

It is the anhydride of creatine.

It is mostly formed from breakdown of creatine phosphate in the body. This process is not catalysed by any enzymes and is irreversible. Creatine-labelled with isotopic 15 N gives creatinine containing same isotopic 15 N.

A large amount in the muscle.

Effects of Creatinine Feeding:

When orally administered nearly 80% is promptly excreted in the urine. Hence, it is considered to be a waste product. It is a no-threshold substance. It is filterd by the glomeruli and is also actively secreted by the tubular cells in the urine. Amount in blood: Normally it is present about 0.7-2.0 mgm per 100 ml. This level is very constant and it is considered to be pathological when its value increases about 2 mgm. Creatinine is also found in bile, sweat and in secretion of stomach and intestine.

About 1.2 -2.0 gm in adult males and 0.8-1.5 gm in adult females are excreted in 24 hours. The amount excreted is remarkably constant for a particular individual. It is related to the muscle bulk and is higher in muscular persons. This stands in great contrast with creatine excretion, which bears no relation to muscular development. The excretion increases during work and exercise but is immediately followed by a fall, so that the daily output remains constant.

Significance of Variation:

Creatinine represents the waste products of creatine metabolism and it arises in the body from the spontaneous breakdown of creation phosphate. It serves practically no function in the body apparently. As its excretion is not related with food protein so its variations in the excretion indicate some of the metabolic disorders. Appearance of creatinine in urine is known as creatinuria when a small amount of creatine is also excreted along with creatinine.

The creatine value gradually decreases as the maturity is advanced. Its excretion increases in fevers, starvation, on a carbohydrate-free diet and in diabetes mellitus. It may increase due to excessive tissue destruction releasing creatine or due to failure of creatine being properly phosphorylated. So creatinine excretion is independent of food proteins and is to be considered as an index of endogenous protein metabolism.


Contents

There are four JAK proteins: JAK1, JAK2, JAK3 and TYK2. [1] JAKs contains a FERM domain (approximately 400 residues), an SH2-related domain (approximately 100 residues), a kinase domain (approximately 250 residues) and a pseudokinase domain (approximately 300 residues). [2] The kinase domain is vital for JAK activity, since it allows JAKs to phosphorylate (add phosphate groups to) proteins.

There are seven STAT proteins: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6. [1] STAT proteins contain many different domains, each with a different function, of which the most conserved region is the SH2 domain. [2] The SH2 domain is formed of 2 α-helices and a β-sheet and is formed approximately from residues 575–680. [2] [3] STATs also have transcriptional activation domains (TAD), which are less conserved and are located at the C-terminus. [4] In addition, STATs also contain: tyrosine activation, amino-terminal, linker, coiled-coil and DNA-binding domains. [4]

The binding of various ligands, usually cytokines, such as interferons and interleukins, to cell-surface receptors, causes the receptors to dimerize, which brings the receptor-associated JAKs into close proximity. [6] The JAKs then phosphorylate each other on tyrosine residues located in regions called activation loops, through a process called transphosphorylation, which increases the activity of their kinase domains. [6] The activated JAKs then phosphorylate tyrosine residues on the receptor, creating binding sites for proteins possessing SH2 domains. [6] STATs then bind to the phosphorylated tyrosines on the receptor using their SH2 domains, and then they are tyrosine-phosphorylated by JAKs, causing the STATs to dissociate from the receptor. [2] At least STAT5 requires glycosylation at threonine 92 for strong STAT5 tyrosine phosphorylation. [7] These activated STATs form hetero- or homodimers, where the SH2 domain of each STAT binds the phosphorylated tyrosine of the opposite STAT, and the dimer then translocates to the cell nucleus to induce transcription of target genes. [2] STATs may also be tyrosine-phosphorylated directly by receptor tyrosine kinases - but since most receptors lack built-in kinase activity, JAKs are usually required for signalling. [1]

Movement of STATs from the cytosol to the nucleus Edit

To move from the cytosol to the nucleus, STAT dimers have to pass through nuclear pore complexes (NPCs), which are protein complexes present along the nuclear envelope that control the flow of substances in and out of the nucleus. To enable STATs to move into the nucleus, an amino acid sequence on STATs, called the nuclear localization signal (NLS), is bound by proteins called importins. [4] Once the STAT dimer (bound to importins) enters the nucleus, a protein called Ran (associated with GTP) binds to the importins, releasing them from the STAT dimer. [8] The STAT dimer is then free in the nucleus.

Specific STATs appear to bind to specific importin proteins. For example, STAT3 proteins can enter the nucleus by binding to importin α3 and importin α6. [9] On the other hand, STAT1 and STAT2 bind to importin α5. [4] Studies indicate that STAT2 requires a protein called interferon regulatory factor 9 (IRF9) to enter the nucleus. [8] Not as much is known about nuclear entrance of other STATs, but it has been suggested that a sequence of amino acids in the DNA-binding domain of STAT4 might allow nuclear import also, STAT5 and STAT6 can both bind to importin α3. [8] In addition, STAT3, STAT5 and STAT6 can enter the nucleus even if they are not phosphorylated at tyrosine residues. [8]

Role of post-translational modifications Edit

After STATs are made by protein biosynthesis, they have non-protein molecules attached to them, called post-translational modifications. One example of this is tyrosine phosphorylation (which is fundamental for JAK-STAT signalling), but STATs experience other modifications, which may affect STAT behaviour in JAK-STAT signalling. These modifications include: methylation, acetylation and serine phosphorylation.

  • Methylation. STAT3 can be dimethylated (have two methyl groups) on a lysine residue, at position 140, and it is suggested that this could reduce STAT3 activity. [10] There is debate as to whether STAT1 is methylated on an arginine residue (at position 31), and what the function of this methylation could be. [11]
  • Acetylation. STAT1, STAT2, STAT3, STAT5 and STAT6 have been shown to be acetylated. [12] STAT1 may have an acetyl group attached to lysines at positions 410 and 413, and as a result, STAT1 can promote the transcription of apoptotic genes - triggering cell death. [12] STAT2 acetylation is important for interactions with other STATs, and for the transcription of anti-viral genes. [4]

Acetylation of STAT3 has been suggested to be important for its dimerization, DNA-binding and gene-transcribing ability, and IL-6 JAK-STAT pathways that use STAT3 require acetylation for transcription of IL-6 response genes. [12] STAT5 acetylation on lysines at positions 694 and 701 is important for effective STAT dimerization in prolactin signalling. [13] Adding acetyl groups to STAT6 is suggested to be essential for gene transcription in some forms of IL-4 signalling, but not all the amino acids which are acetylated on STAT6 are known. [12]

  • Serine phosphorylation. Most of the seven STATs (except STAT2) undergo serine phosphorylation. [2] Serine phosphorylation of STATs has been shown to reduce gene transcription. [14] It is also required for the transcription of some target genes of the cytokines IL-6 and IFN- γ. [11] It has been proposed that phosphorylation of serine can regulate STAT1 dimerization, [11] and that continuous serine phosphorylation on STAT3 influences cell division. [15]

Recruitment of co-activators Edit

Like many other transcription factors, STATs are capable of recruiting co-activators such as CBP and p300, and these co-activators increase the rate of transcription of target genes. [2] The coactivators are able to do this by making genes on DNA more accessible to STATs and by recruiting proteins needed for transcription of genes. The interaction between STATs and coactivators occurs through the transactivation domains (TADs) of STATs. [2] The TADs on STATs can also interact with histone acetyltransferases (HATs) [16] these HATs add acetyl groups to lysine residues on proteins associated with DNA called histones. Adding acetyl groups removes the positive charge on lysine residues, and as a result there are weaker interactions between histones and DNA, making DNA more accessible to STATs and enabling an increase in the transcription of target genes.

Integration with other signalling pathways Edit

JAK-STAT signalling is able to interconnect with other cell-signalling pathways, such as the PI3K/AKT/mTOR pathway. [17] When JAKs are activated and phosphorylate tyrosine residues on receptors, proteins with SH2 domains (such as STATs) are able bind to the phosphotyrosines, and the proteins can carry out their function. Like STATs, the PI3K protein also has an SH2 domain, and therefore it is also able to bind to these phosphorylated receptors. [17] As a result, activating the JAK-STAT pathway can also activate PI3K/AKT/mTOR signalling.

JAK-STAT signalling can also integrate with the MAPK/ERK pathway. Firstly, a protein important for MAPK/ERK signalling, called Grb2, has an SH2 domain, and therefore it can bind to receptors phosphorylated by JAKs (in a similar way to PI3K). [17] Grb2 then functions to allow the MAPK/ERK pathway to progress. Secondly, a protein activated by the MAPK/ERK pathway, called MAPK (mitogen-activated protein kinase), can phosphorylate STATs, which can increase gene transcription by STATs. [17] However, although MAPK can increase transcription induced by STATs, one study indicates that phosphorylation of STAT3 by MAPK can reduce STAT3 activity. [18]

One example of JAK-STAT signalling integrating with other pathways is Interleukin-2 (IL-2) receptor signaling in T cells. IL-2 receptors have γ (gamma) chains, which are associated with JAK3, which then phosphorylates key tyrosines on the tail of the receptor. [19] Phosphorylation then recruits an adaptor protein called Shc, which activates the MAPK/ERK pathway, and this facilitates gene regulation by STAT5. [19]

Alternative signalling pathway Edit

An alternative mechanism for JAK-STAT signalling has also been suggested. In this model, SH2 domain-containing kinases, can bind to phosphorylated tyrosines on receptors and directly phosphorylate STATs, resulting in STAT dimerization. [6] Therefore, unlike the traditional mechanism, STATs can be phosphorylated not just by JAKs, but by other receptor-bound kinases. So, if one of the kinases (either JAK or the alternative SH2-containing kinase) cannot function, signalling may still occur through activity of the other kinase. [6] This has been shown experimentally. [20]

Given that many JAKs are associated with cytokine receptors, the JAK-STAT signalling pathway plays a major role in cytokine receptor signalling. Since cytokines are substances produced by immune cells that can alter the activity of neighbouring cells, the effects of JAK-STAT signalling are often more highly seen in cells of the immune system. For example, JAK3 activation in response to IL-2 is vital for lymphocyte development and function. [21] Also, one study indicates that JAK1 is needed to carry out signalling for receptors of the cytokines IFNγ, IL-2, IL-4 and IL-10. [22]

The JAK-STAT pathway in cytokine receptor signalling can activate STATs, which can bind to DNA and allow the transcription of genes involved in immune cell division, survival, activation and recruitment. For example, STAT1 can enable the transcription of genes which inhibit cell division and stimulate inflammation. [2] Also, STAT4 is able to activate NK cells (natural killer cells), and STAT5 can drive the formation of white blood cells. [2] [23] In response to cytokines, such as IL-4, JAK-STAT signalling is also able to stimulate STAT6, which can promote B-cell proliferation, immune cell survival, and the production of an antibody called IgE. [2]

JAK-STAT signalling plays an important role in animal development. The pathway can promote blood cell division, as well as differentiation (the process of a cell becoming more specialised). [24] In some flies with faulty JAK genes, too much blood cell division can occur, potentially resulting in leukaemia. [25] JAK-STAT signalling has also been associated with excessive white blood cell division in humans and mice. [24]

The signalling pathway is also crucial for eye development in the fruit fly (Drosophila melanogaster). When mutations occur in genes coding for JAKs, some cells in the eye may be unable to divide, and other cells, such as photoreceptor cells, have been shown not to develop correctly. [24]

The entire removal of a JAK and a STAT in Drosophila causes death of Drosophila embryos, whilst mutations in the genes coding for JAKs and STATs can cause deformities in the body patterns of flies, particularly defects in forming body segments. [24] One theory as to how interfering with JAK-STAT signalling might cause these defects is that STATs may directly bind to DNA and promote the transcription of genes involved in forming body segments, and therefore by mutating JAKs or STATs, flies experience segmentation defects. [26] STAT binding sites have been identified on one of these genes, called even-skipped (eve), to support this theory. [27] Of all the segment stripes affected by JAK or STAT mutations, the fifth stripe is affected the most, the exact molecular reasons behind this are still unknown. [24]

Given the importance of the JAK-STAT signalling pathway, particularly in cytokine signalling, there are a variety of mechanisms that cells possess to regulate the amount of signalling that occurs. Three major groups of proteins that cells use to regulate this signalling pathway are protein inhibitors of activated STAT (PIAS), [28] protein tyrosine phosphatases (PTPs) [29] and suppressors of cytokine signalling (SOCS). [30]

Protein inhibitors of activated STATs (PIAS) Edit

PIAS are a four-member protein family made of: PIAS1, PIAS3, PIASx, and PIASγ. [31] The proteins add a marker, called SUMO (small ubiquitin-like modifier), onto other proteins – such as JAKs and STATs, modifying their function. [31] The addition of a SUMO group onto STAT1 by PIAS1 has been shown to prevent activation of genes by STAT1. [32] Other studies have demonstrated that adding a SUMO group to STATs may block phosphorylation of tyrosines on STATs, preventing their dimerization and inhibiting JAK-STAT signalling. [33] PIASγ has also been shown to prevent STAT1 from functioning. [34] PIAS proteins may also function by preventing STATs from binding to DNA (and therefore preventing gene activation), and by recruiting proteins called histone deacetylases (HDACs), which lower the level of gene expression. [31]

Protein tyrosine phosphatases (PTPs) Edit

Since adding phosphate groups on tyrosines is such an important part of how the JAK-STAT signalling pathway functions, removing these phosphate groups can inhibit signalling. PTPs are tyrosine phosphatases, so are able to remove these phosphates and prevent signalling. Three major PTPs are SHP-1, SHP-2 and CD45. [35]

    . SHP-1 is mainly expressed in blood cells. [36] It contains two SH2 domains and a catalytic domain (the region of a protein that carries out the main function of the protein) - the catalytic domain contains the amino acid sequence VHCSAGIGRTG (a sequence typical of PTPs). [37] As with all PTPs, a number of amino acid structures are essential for their function: conserved cysteine, arginine and glutamine amino acids, and a loop made of tryptophan, proline and aspartate amino acids (WPD loop). [37] When SHP-1 is inactive, the SH2 domains interact with the catalytic domain, and so the phosphatase is unable to function. [37] When SHP-1 is activated however, the SH2 domains move away from the catalytic domain, exposing the catalytic site and therefore allowing phosphatase activity. [37] SHP-1 is then able to bind and remove phosphate groups from the JAKs associated with receptors, preventing the transphosphorylation needed for the signalling pathway to progress.

One example of this is seen in the JAK-STAT signalling pathway mediated by the erythropoietin receptor (EpoR). Here, SHP-1 binds directly to a tyrosine residue (at position 429) on EpoR and removes phosphate groups from the receptor-associated JAK2. [38] The ability of SHP-1 to negatively regulate the JAK-STAT pathway has also been seen in experiments using mice lacking SHP-1. [39] These mice experience characteristics of autoimmune diseases and show high levels of cell proliferation, which are typical characteristics of an abnormally high level of JAK-STAT signalling. [39] Additionally, adding methyl groups to the SHP-1 gene (which reduces the amount of SHP-1 produced) has been linked to lymphoma (a type of blood cancer) . [40]

However, SHP-1 may also promote JAK-STAT signalling. A study in 1997 found that SHP-1 potentially allows higher amounts of STAT activation, as opposed to reducing STAT activity. [41] A detailed molecular understanding for how SHP-1 can both activate and inhibit the signalling pathway is still unknown. [35]

    . SHP-2 has a very similar structure to SHP-1, but unlike SHP-1, SHP-2 is produced in many different cell types - not just blood cells. [42] Humans have two SHP-2 proteins, each made up of 593 and 597 amino acids. [37] The SH2 domains of SHP-2 appear to play an important role in controlling the activity of SHP-2. One of the SH2 domains binds to the catalytic domain of SHP-2, to prevent SHP-2 functioning. [35] Then, when a protein with a phosphorylated tyrosine binds, the SH2 domain changes orientation and SHP-2 is activated. [35] SHP-2 is then able to remove phosphate groups from JAKs, STATs and the receptors themselves - so, like SHP-1, can prevent the phosphorylation needed for the pathway to continue, and therefore inhibit JAK-STAT signalling. Like SHP-1, SHP-2 is able to remove these phosphate groups through the action of the conserved cysteine, arginine, glutamine and WPD loop. [37]

Negative regulation by SHP-2 has been reported in a number of experiments - one example has been when exploring JAK1/STAT1 signalling, where SHP-2 is able to remove phosphate groups from proteins in the pathway, such as STAT1. [43] In a similar manner, SHP-2 has also been shown to reduce signalling involving STAT3 and STAT5 proteins, by removing phosphate groups. [44] [45]

Like SHP-1, SHP-2 is also believed to promote JAK-STAT signalling in some instances, as well as inhibit signalling. For example, one study indicates that SHP-2 may promote STAT5 activity instead of reducing it. [46] Also, other studies propose that SHP-2 may increase JAK2 activity, and promote JAK2/STAT5 signalling. [47] It is still unknown how SHP2 can both inhibit and promote JAK-STAT signalling in the JAK2/STAT5 pathway one theory is that SHP-2 may promote activation of JAK2, but inhibit STAT5 by removing phosphate groups from it. [35]

    . CD45 is mainly produced in blood cells. [4] In humans it has been shown to be able to act on JAK1 and JAK3, [48] whereas in mice, CD45 is capable of acting on all JAKs. [49] One study indicates that CD45 can reduce the amount of time that JAK-STAT signalling is active. [49] The exact details of how CD45 functions is still unknown. [35]

Suppressors of cytokine signalling (SOCS) Edit

There are eight protein members of the SOCS family: cytokine-inducible SH2 domain-containing protein (CISH), SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS7, each protein has an SH2 domain and a 40-amino-acid region called the SOCS box. [50] The SOCS box can interact with a number of proteins to form a protein complex, and this complex can then cause the breakdown of JAKs and the receptors themselves, therefore inhibiting JAK-STAT signalling. [4] The protein complex does this by allowing a marker called ubiquitin to be added to proteins, in a process called ubiquitination, which signals for a protein to be broken down. [51] The proteins, such as JAKs and the receptors, are then transported to a compartment in the cell called the proteasome, which carries out protein breakdown. [51]

SOCS can also function by binding to proteins involved in JAK-STAT signalling and blocking their activity. For example, the SH2 domain of SOCS1 binds to a tyrosine in the activation loop of JAKs, which prevents JAKs from phosphorylating each other. [4] The SH2 domains of SOCS2, SOCS3 and CIS bind directly to receptors themselves. [51] Also, SOCS1 and SOCS3 can prevent JAK-STAT signalling by binding to JAKs, using segments called kinase inhibitory regions (KIRs) and stopping JAKs binding to other proteins. [52] The exact details of how other SOCS function is less understood. [4]

Regulator Positive or Negative regulation Function
PTPs SHP-1 and SHP-2: Negative, but could also be positive. CD45, PTP1B, TC-PTP: Negative Removes phosphate groups from receptors, JAKs and STATs
SOCS Negative SOCS1 and SOCS3 block JAKs active sites using KIR domains. SOCS2, SOCS3 and CIS can bind receptors. SOCS1 and SOCS3 can signal JAKs and receptor for degradation.
PIAS Negative Add SUMO group to STATs to inhibit STAT activity. Recruit histone deacetylases to lower gene expression. Prevent STATs binding to DNA.

Since the JAK-STAT pathway plays a major role in many fundamental processes, such as apoptosis and inflammation, dysfunctional proteins in the pathway may lead to a number of diseases. For example, alterations in JAK-STAT signalling can result in cancer and diseases affecting the immune system, such as severe combined immunodeficiency disorder (SCID). [53]

Immune system-related diseases Edit

JAK3 can be used for the signalling of IL-2, IL-4, IL-15 and IL-21 (as well as other cytokines) therefore patients with mutations in the JAK3 gene often experience issues affecting many aspects of the immune system. [54] [55] For example, non-functional JAK3 causes SCID, which results in patients having no NK cells, B cells or T cells, and this would make SCID individuals susceptible to infection. [55] Mutations of the STAT5 protein, which can signal with JAK3, has been shown to result in autoimmune disorders. [56]

It has been suggested that patients with mutations in STAT1 and STAT2 are often more likely to develop infections from bacteria and viruses. [57] Also, STAT4 mutations have been associated with rheumatoid arthritis, and STAT6 mutations are linked to asthma. [58] [59]

Patients with a faulty JAK-STAT signalling pathway may also experience skin disorders. For example, non-functional cytokine receptors, and overexpression of STAT3 have both been associated with psoriasis (an autoimmune disease associated with red, flaky skin). [55] STAT3 plays an important role in psoriasis, as STAT3 can control the production of IL-23 receptors, and IL-23 can help the development of Th17 cells, and Th17 cells can induce psoriasis. [60] Also, since many cytokines function through the STAT3 transcription factor, STAT3 plays a significant role in maintaining skin immunity. [55] In addition, because patients with JAK3 gene mutations have no functional T cells, B cells or NK cells, they would more likely to develop skin infections.

Cancer Edit

Cancer involves abnormal and uncontrollable cell growth in a part of the body. Therefore, since JAK-STAT signalling can allow the transcription of genes involved in cell division, one potential effect of excessive JAK-STAT signalling is cancer formation. High levels of STAT activation have been associated with cancer in particular, high amounts of STAT3 and STAT5 activation is mostly linked to more dangerous tumours. [61] For example, too much STAT3 activity has been associated with increasing the likelihood of melanoma (skin cancer) returning after treatment and abnormally high levels of STAT5 activity have been linked to a greater probability of patient death from prostate cancer. [62] [61] Altered JAK-STAT signalling can also be involved in developing breast cancer. JAK-STAT signalling in mammary glands (located within breasts) can promote cell division and reduce cell apoptosis during pregnancy and puberty, and therefore if excessively activated, cancer can form. [63] High STAT3 activity plays a major role in this process, as it can allow the transcription of genes such as BCL2 and c-Myc, which are involved in cell division. [63]

Mutations in JAK2 can lead to leukaemia and lymphoma. [6] Specifically, mutations in exons 12, 13, 14 and 15 of the JAK2 gene are proposed to be a risk factor in developing lymphoma or leukemia. [6] Additionally, mutated STAT3 and STAT5 can increase JAK-STAT signalling in NK and T cells, which promotes very high proliferation of these cells, and increases the likelihood of developing leukaemia. [63] Also, a JAK-STAT signalling pathway mediated by erythropoietin (EPO), which usually allows the development of red blood cells, may be altered in patients with leukemia. [64]

Treatments Edit

Since excessive JAK-STAT signalling is responsible for some cancers and immune disorders, JAK inhibitors have been proposed as drugs for therapy. For instance, to treat some forms of leukaemia, targeting and inhibiting JAKs could eliminate the effects of EPO signalling and perhaps prevent the development of leukaemia. [64] One example of a JAK inhibitor drug is Ruxolitinib, which is used as a JAK2 inhibitor. [61] STAT inhibitors are also being developed, and many of the inhibitors target STAT3. [63] It has been reported that therapies which target STAT3 can improve the survival of patients with cancer. [63] Another drug, called Tofacitinib, has been used for psoriasis and rheumatoid arthritis treatment, and has been recently approved for Crohn's Disease and Ulcerative Colitis treatment. [53]


Imaging and Spectroscopic Analysis of Living Cells

Xin Zhou , . Jin Zhang , in Methods in Enzymology , 2012

3.1.1 Introduction

Protein kinases are enzymes that catalyze the transfer of the γ-phosphate of ATP to the protein substrates, thus altering their functions. These signaling enzymes play a critical and complex role in regulating cellular signal transduction as they orchestrate multiple intracellular processes such as glycogen synthesis, hormone responses, and ion transport. Many signaling cascades involving protein kinases require dynamic control and spatial compartmentalization of kinase activity, which needs to be tracked continuously in different compartments and signaling microdomains in living cells however, traditional methods to study protein kinases activity only provide static and limited snapshots of signaling events and fail to capture the dynamic changes of kinase activity. On the other hand, genetically encodable FRET-based biosensors offer a versatile and powerful approach to elucidate the spatiotemporal patterns of kinase signaling.


PHOSPHORYLATION - (Nov/19/2006 )

Assuming the size is 57 KD, then phosphorylation will have little or no effect on the position of the band on an SDS PAGE gel. SDS linearizes the protein and makes the electrophoresis essentially independent of charge, and phosphorylation changes the molecular weight of such a large protein by only a very small amount.

it depends of on the extensity of phosphorylation about 12 mol Pi per mol protein make a shift of about 1 kDa there are papers where scientist discriminate autophosphorylation forms of kinases by 1 to 2 kDa in appropriate SDS systems many proteins are multiple phosphorylatable even at non-documented phosphorylation sites

If you run a long sds-page gel and use antibody against phosphorylated and unphosphorylated form of the protein, you should be able to see the two forms on the blot. But whenever I try to differentiate phosphorylated and unphosphorylated Rb on the western blot I am get more than two bands.

If you run a long sds-page gel and use antibody against phosphorylated and unphosphorylated form of the protein, you should be able to see the two forms on the blot. But whenever I try to differentiate phosphorylated and unphosphorylated Rb on the western blot I am get more than two bands.

many if not most proteins are multiple phosphorylated in SDS only S/T and Y-phosphorylations are stable each S/T or Y is a potential phosphorylation site despite if it is documented as a genuine phosphorylation site or not

so multiple bands may represent different states/degrees of phosphorylation

if you are really interested in degree of phosphorylation, try to correlate pI and phosphorylation if you know the native pI

nowadays, i am trying to figure out how poeple assign to one protein i am working on that has molecular weight of 62KDa, and maximum of three phosphorylation sites, a 72KDa form to be its phosphorylated form. I am trying to prove that it is not its phosphorylated form simply because it can't be !!

! so up to you unphosphorylated and phosphorylated proteins (max of three sites) cant be distinguished on simple 1D SDS-PAGE.

yes
by using the specific antibody for ur phosphorylation sites.

! so up to you unphosphorylated and phosphorylated proteins (max of three sites) cant be distinguished on simple 1D SDS-PAGE.


Binding Initiates a Signaling Pathway

After the ligand binds to the cell-surface receptor, the activation of the receptor&rsquos intracellular components sets off a chain of events that is called a signaling pathway or a signaling cascade. In a signaling pathway, second messengers, enzymes, and activated proteins interact with specific proteins, which are in turn activated in a chain reaction that eventually leads to a change in the cell&rsquos environment (Figure (PageIndex<1>)). The events in the cascade occur in a series, much like a current flows in a river. Interactions that occur before a certain point are defined as upstream events, and events after that point are called downstream events.

Art Connection Figure (PageIndex<1>): The epidermal growth factor (EGF) receptor (EGFR) is a receptor tyrosine kinase involved in the regulation of cell growth, wound healing, and tissue repair. When EGF binds to the EGFR, a cascade of downstream events causes the cell to grow and divide. If EGFR is activated at inappropriate times, uncontrolled cell growth (cancer) may occur.

In certain cancers, the GTPase activity of the RAS G-protein is inhibited. This means that the RAS protein can no longer hydrolyze GTP into GDP. What effect would this have on downstream cellular events?

Signaling pathways can get very complicated very quickly because most cellular proteins can affect different downstream events, depending on the conditions within the cell. A single pathway can branch off toward different endpoints based on the interplay between two or more signaling pathways, and the same ligands are often used to initiate different signals in different cell types. This variation in response is due to differences in protein expression in different cell types. Another complicating element is signal integration of the pathways, in which signals from two or more different cell-surface receptors merge to activate the same response in the cell. This process can ensure that multiple external requirements are met before a cell commits to a specific response.

The effects of extracellular signals can also be amplified by enzymatic cascades. At the initiation of the signal, a single ligand binds to a single receptor. However, activation of a receptor-linked enzyme can activate many copies of a component of the signaling cascade, which amplifies the signal.


7 Ways to Study Protein Phosphorylation

2%) by a kinase and removed by a phosphatase (Figure 1). [1] Phosphorylation at other amino acids have also been reported. [2] Phosphorylation can modify protein structure, function, and interactions. As such, phosphorylation plays a critical role in virtually all cellular processes in homeostasis and disease, including signal transduction, cell cycle, differentiation, proliferation, metabolism, motility, and death. [3,4] Importantly, phosphorylation at different residues can cause different outcomes. For example, RAF1 is a kinase central to the MAPK pathway that is activated when it is phosphorylated at serine (S) or threonine (T) residues S259, S338, S340/341, T491, or S494. [5,6] However, phosphorylation at S289/296/301 results in the inhibition of RAF1 kinase activity. [7] Understanding the specific sites and level of phosphorylation is paramount in understanding cell signaling and phenotype. In this blog, seven research tools for studying protein phosphorylation are discussed and compared.

Figure 1. Addition and removal of protein phosphorylation via kinases and phosphatases.

Research Methods

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Proteins in a sample – often cell or tissue lysates – are first separated by size using SDS-PAGE. This is possible because the negatively-charged SDS in the buffer and gel applies a negative charge to all amino acids uniformly, enabling the separation of the proteins in an electric field based on size rather than by their unique amino acid content and charge (Figure 2A). [8] Proteins can then be visualized with Coomassie blue or silver staining. Notably, SDS-PAGE with Coomassie blue or silver staining can be used to detect some phosphorylation events that cause proteins to migrate slower than their unphosphorylated counterparts. A clear disadvantage to this approach is that migration shifts due to other factors (e.g., glycosylation) cannot be eliminated. Since all proteins will be stained with this approach, the analysis of purified proteins is primarily performed. Including the appropriate controls is an important consideration (see “Experimental Controls” box).

Figure 2. Western blotting procedure to detect phosphorylated proteins. A) Proteins are separated via SDS-PAGE where lane 1 = protein ladder, lane 2 = unphosphorylated protein, and lane 3 = phosphorylated protein. B) Proteins are transferred to a membrane via voltage. C) The membrane is incubated with an antibody specific to the phosphorylated site-of-interest and then an HRP-conjugated secondary antibody. D) After an HRP substrate is added, antibody binding (to the band representing the phosphorylated protein) is detected via chemiluminescence.
Experimental Controls

Including the right controls is essential to every well-performed experiment, and phosphorylation studies are no different. Important controls to consider include:

  • Expression level of a housekeeping gene (e.g., GAPDH, beta-actin) to ensure that the same amount of protein is added to each well of an SDS-PAGE gel (loading control for western blotting).
  • Expression level of the total protein (i.e., both phosphorylated and unphosphorylated protein). This will help determine whether protein phosphorylation or expression of the protein is up- or down-regulated.
  • Phosphorylated peptide or protein with phosphorylation at a specific site (positive control). Peptides can be synthesized chemically. Phosphorylated proteins may be obtained 1) with in vitro phosphorylation by mixing a kinase with known activity with the protein-of-interest, or 2) by stimulating cells with known effect on the protein-of-interest.
  • Unphosphorylated peptide or protein (negative control). In vitro kinase assays can either be performed without ATP or the phosphorylated proteins can be de-phosphorylated using phosphatases, such as lambda protein phosphatase. For cell culture experiments, an unphosphorylated control may be obtained with cells that have 1) not been stimulated or 2) been treated with a specific kinase inhibitor.
  • Unlabeled cells to determine gating parameters (negative control for flow cytometry)
  • Isotype control to determine gating parameters. An isotype control is an antibody with the same fluorophore, but not specific to the protein-of-interest (negative control for flow cytometry)

Please note that the types of controls may vary based on the research tool that is employed. Additional controls not listed above may be necessary.

A more traditional approach to detect phosphorylated proteins using SDS-PAGE employs radio-labeled ATP during kinase reactions. The phosphorylated protein is then detected via its incorporated radio-labeled ATP with autoradiography, fluorography, or phosphor imaging. [8] Unlike Coomassie or silver staining, the use of radio-labeled ATP enables the specific detection of unpurified phosphorylated proteins. Unfortunately, the radioactive label may not be amenable for all phosphorylation events and it poses health hazards. Alternatives to using radio-labeled ATP to detect phosphorylated proteins include staining with methyl green after phosphoester bond hydrolysis, using a fluorescent dye that specifically binds to phosphoryl groups (e.g., Pro-Q Diamond), or using metal chelation to conjugate a dye to the phosphoryl group. [9] For example, Phos-Tag ™ is a dinuclear metal complex that binds strongly to the phosphoryl group at neutral pH. [10] Phos-Tag ™ is precast into a gel and significantly inhibits the migration of phosphorylated proteins regardless of the phosphorylation site. Thus, any phosphorylation will result in a slower migration than the unphosphorylated protein, and a protein with several phosphorylation events may result in multiple bands that are easily discerned from one another. Purified proteins can be analyzed using Coomassie or silver staining, whereas unpurified proteins can be analyzed with western blotting (see next section). Phos-Tag ™ can be used in conjunction with other applications (e.g., western blotting, mass spectrometry) to detect phosphorylated proteins.

Relative differences in band intensities across different sample treatments with SDS-PAGE are compared by eye, but densitometry signal can be extracted to obtain semi-quantitative data. Quantitative data is possible with SDS-PAGE analysis using a standard curve, but is not usually performed. [11]



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