This suggests that serine phosphorylation alone is a means of priming STATs for an altered transcriptional response once a second stimulus causing tyrosine phosphorylation is received. From the current literature we would infer primed STATs to cause increased transcription in most cases, but the negative effect reported for STAT3 complicates the issue and demands further clarification. Therefore, it is not yet possible to judge the importance of serine phosphorylation for STAT3-dependent responses, e.
In spite of this, recent data support the notion that STAT3 serine phosphorylation is important for biological responses. STAT3 is constitutively phosphorylated in v-Src-transformed cells and participates in v-Src-mediated fibroblast transformation Bromberg et al. Furthermore, inhibition of STAT3 serine phosphorylation resulted in reduced transformation by the v- src , but not the v- ras oncogene Turkson et al.
Taken together, these findings suggest an important role for STAT3 and the phosphorylation of S in cellular growth control. Uncovering the relevance of serine phosphorylation for cellular responses or the integrity of organisms is a major challenge for future research. Indications as to which signaling pathways target the STAT P M SP motif were first and mostly obtained using more or less specific inhibitors of serine kinases. As will be described below, pharmacological studies were in some cases complemented by experiments with dominant-negative, constitutively active, or inhibitor-resistant kinase alleles, as well as with in-vitro phosphorylation assays Table 2.
Before proceeding to a more detailed description of individual signaling routes we would like to point out the following more general aspects of the relationship between signal transducers and STAT serine phosphorylation: 1 Serine phosphorylation of one STAT can occur through different signal transduction paths. This probably applies for those phosphorylation events, which are sensitive to H7, but insensitive to PD and SB The enzymatic parameters for the reaction between a candidate kinase and a STAT substrate should be comparable to those obtained with known in-vivo substrates and a tight correlation should exist between activation of a candidate kinase and STAT phosphorylation in vivo.
Inconsistent results have been obtained with different experimental protocols concerning the phosphorylation of STATs by different MAPK in-vitro or with regard to the effects of particular signaling pathways on serine phosphorylation of, and transcriptional activation by STATS [e.
Lim and Cao and Schuringa et al. Inhibition of phosphorylation by PD is thought to be a quite specific criterion for an involvement of the ERK pathway and this compound interferes with STAT3 serine phosphorylation by receptor tyrosine kinases, IL-2, and lymphocyte antigen receptors Chung et al.
We would again like to stress the fact that this is due to Sdependent and independent down-regulation of tyrosine phosphorylation and does not indicate a negative effect of phospho-S per se on STAT3-mediated transcription. While these data support a role for ERKs as STAT1 serine kinases, several lines of evidence argue against a general validity of the concept.
It is also puzzling that the study by Schuringa et al. An involvement of the kinase is further suggested by transfection experiments employing dominant-negative or constitutively active p38MAPK kinase MKK6, Goh et al. This may indicate intermediate steps or a need for relocation of p38MAPK from the nucleus to the cytoplasm.
By contrast, Goh et al. Goh et al. Yokogami et al. The mTor kinase, a phosphatidylinositol kinase-related enzyme, is sensitive to rapamycin treatment. Consistent with this result, Ramana et al. If physiologically relevant these results suggest that permanent PP2A activity be required to keep STATs dephosphorylated on S and that an increase in phosphorylation demands the inhibition of this phosphatase.
However, our knowledge of STAT serine dephosphorylation is at a very preliminary stage. Therefore, considerable information has been gathered about biological stimuli that cause serine phosphorylation. While it is clear at least in some cases that serine phosphorylation influences the transcriptional activity of STATs, the mechanisms by which this occurs, as well as the biological impact it causes, are unclear in most situations. Signal transduction pathways are beginning to emerge, but much more needs to be learned about the kinases phosphorylating STATs on serine in different situations.
This review is a snapshot documenting the current situation in a dynamic process aimed at providing more definitive answers about causes and effects of STAT serine phosphorylation. Darnell Jr JE.
Decker T and Kovarik P. Hilton DJ. Phosphorylation is the most common and important molecular mechanism of acute and reversible regulation of protein function. Through protein phosphorylation, protein function is regulated in response to extracellular stimuli both inside and outside the cell. Protein phosphorylation is usually analyzed by biosynthetic labeling with 32 P-labeled inorganic phosphate 32 P i , and the studies of mammalian cells metabolically labeled with 32 P orthophosphate suggest that as many as one-third of all cellular proteins are covalently modified by protein phosphorylation.
Most proteins are found to be phosphorylated at serine or threonine residues, and many proteins involved in signal transduction are also phosphorylated at tyrosine residues. Due to a large number of kinases and phosphatases in the genome, the identification of the specific enzymes responsible for a given site in a given protein is immensely challenging.
However, because protein kinases and phosphatases recognize local specificity determinants within proteins, it is possible to use small peptides to study the characteristics of site-specific phosphorylation. In addition, phosphorylation usually causes retardation in gel mobility, providing an opportunity to investigate peptide phosphorylation and de-phosphorylation by monitoring migration on high-resolution peptide gels.
Phosphorylation acts as a molecular switch to directly turn on or off the functions of proteins, or it may more subtly regulate their functions, for example by controlling their locations inside of cells, interactions with other proteins, or their degradation by proteases. The phosphate group that is transferred onto proteins by protein kinases is removed from the high energy compound adenosine-triphosphate ATP , which fuels most chemical reactions in the body.
Research conducted by Kinexus is supporting the novel hypothesis that the predominant function of hyperphosphorylation is to mediate the destruction of proteins. Phosphorylation is found most commonly on specific serine and threonine amino acid residues in proteins, but it also occurs on tyrosine and other amino acid residues histidine, aspartic acid, glutamic acid as well.
Therefore, protein-tyrosine phosphorylation levels appear to be about times lower than protein-serine phosphorylation levels and times lower than protein-threonine phosphorylation levels. In cells like activated platelets, tyrosine phosphorylation can be orders of magnitude higher.
This difference in total amount of protein-tyrosine phosphorylation and the frequency of occurrence of tyrosine phosphorylation sites in proteins appears to reflect higher levels of tyrosine phosphorylation sites in lower abundance proteins such as protein kinases. Kinexus has now performed a careful analysis of over , known human phosphorylation sites. Based on the number of known tyrosine phosphorylation sites and their relative occurrence, we conclude that there is likely to be over a million human phosphorylation sites.
The predominant phosphorylation sites were found on the cytoplasmic domain. RP-HPLC analyses of the tryptic peptides of whole band 3 protein, and of the isolated cytoplasmic and membrane-spanning domains allowed for the precise localization of the phosphorylated residues. In this region, all the residues susceptible to phosphorylation were labeled but in varying proportion.
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