Identifying Human Kinase-Specific Protein Phosphorylation Sites by Integrating Heterogeneous Information from Various Sources

Phosphorylation is an important type of protein post-translational modification. Identification of possible phosphorylation sites of a protein is important for understanding its functions. Unbiased screening for phosphorylation sites by in vitro or in vivo experiments is time consuming and expensive; in silico prediction can provide functional candidates and help narrow down the experimental efforts. Most of the existing prediction algorithms take only the polypeptide sequence around the phosphorylation sites into consideration. However, protein phosphorylation is a very complex biological process in vivo. The polypeptide sequences around the potential sites are not sufficient to determine the phosphorylation status of those residues. In the current work, we integrated various data sources such as protein functional domains, protein subcellular location and protein-protein interactions, along with the polypeptide sequences to predict protein phosphorylation sites. The heterogeneous information significantly boosted the prediction accuracy for some kinase families. To demonstrate potential application of our method, we scanned a set of human proteins and predicted putative phosphorylation sites for Cyclin-dependent kinases, Casein kinase 2, Glycogen synthase kinase 3, Mitogen-activated protein kinases, protein kinase A, and protein kinase C families (avaiable at http://cmbi.bjmu.edu.cn/huphospho). The predicted phosphorylation sites can serve as candidates for further experimental validation. Our strategy may also be applicable for the in silico identification of other post-translational modification substrates

Evidence for a Minimal Eukaryotic Phosphoproteome?

BACKGROUND: Reversible phosphorylation catalysed by kinases is probably the most important regulatory mechanism in eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We studied the in vitro phosphorylation of peptide arrays exhibiting the majority of PhosphoBase-deposited protein sequences, by factors in cell lysates from representatives of various branches of the eukaryotic species. We derived a set of substrates from the PhosphoBase whose phosphorylation by cellular extracts is common to the divergent members of different kingdoms and thus may be considered a minimal eukaryotic phosphoproteome. The protein kinases (or kinome) responsible for phosphorylation of these substrates are involved in a variety of processes such as transcription, translation, and cytoskeletal reorganisation. CONCLUSIONS/SIGNIFICANCE: These results indicate that the divergence in eukaryotic kinases is not reflected at the level of substrate phosphorylation, revealing the presence of a limited common substrate space for kinases in eukaryotes and suggests the presence of a set of kinase substrates and regulatory mechanisms in an ancestral eukaryote that has since remained constant in eukaryotic life

Detection and characterization of 3D-signature phosphorylation site motifs and their contribution towards improved phosphorylation site prediction in proteins

<p>Abstract</p> <p>Background</p> <p>Phosphorylation of proteins plays a crucial role in the regulation and activation of metabolic and signaling pathways and constitutes an important target for pharmaceutical intervention. Central to the phosphorylation process is the recognition of specific target sites by protein kinases followed by the covalent attachment of phosphate groups to the amino acids serine, threonine, or tyrosine. The experimental identification as well as computational prediction of phosphorylation sites (P-sites) has proved to be a challenging problem. Computational methods have focused primarily on extracting predictive features from the local, one-dimensional sequence information surrounding phosphorylation sites.</p> <p>Results</p> <p>We characterized the spatial context of phosphorylation sites and assessed its usability for improved phosphorylation site predictions. We identified 750 non-redundant, experimentally verified sites with three-dimensional (3D) structural information available in the protein data bank (PDB) and grouped them according to their respective kinase family. We studied the spatial distribution of amino acids around phosphorserines, phosphothreonines, and phosphotyrosines to extract signature 3D-profiles. Characteristic spatial distributions of amino acid residue types around phosphorylation sites were indeed discernable, especially when kinase-family-specific target sites were analyzed. To test the added value of using spatial information for the computational prediction of phosphorylation sites, Support Vector Machines were applied using both sequence as well as structural information. When compared to sequence-only based prediction methods, a small but consistent performance improvement was obtained when the prediction was informed by 3D-context information.</p> <p>Conclusion</p> <p>While local one-dimensional amino acid sequence information was observed to harbor most of the discriminatory power, spatial context information was identified as relevant for the recognition of kinases and their cognate target sites and can be used for an improved prediction of phosphorylation sites. A web-based service (Phos3D) implementing the developed structure-based P-site prediction method has been made available at <url>http://phos3d.mpimp-golm.mpg.de</url>.</p

Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to predict kinase substrate preferences from the primary structure, hampering the understanding of kinase function in physiology and prompting the development of technologies that allow easy assessment of kinase substrate consensus sequences. Hence, we decided to explore the usefulness of phosphorylation of peptide arrays comprising of 1176 different peptide substrates with recombinant kinases for determining kinase substrate preferences, based on the contribution of individual amino acids to total array phosphorylation. Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology. The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes. Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events

Phosphorylation of Two Serine Residues Regulates Human T-Cell Leukemia Virus Type 2 Rex Function

The function of the human T-cell leukemia virus (HTLV) Rex phosphoprotein is to increase the level of the viral structural and enzymatic gene products expressed from the incompletely spliced viral RNAs containing the Rex-responsive element. The phosphorylation of HTLV type 2 Rex (Rex-2), predominantly on serine residues, correlates with an altered conformation, as detected by a gel mobility shift, and is required for specific binding to its viral RNA target sequence. Thus, the phosphorylation state of Rex in the infected cell may be a switch that determines whether the virus exists in a latent or a productive state. A mutational analysis of Rex-2 that focused on serine and threonine residues was performed to identify regions or domains within Rex-2 important for function, with a specific emphasis on identifying Rex-2 phosphorylation mutants. We identified mutations near the carboxy terminus that disrupted a novel region or domain and abrogated Rex-2 function. Mutant M17 (with S151A and S153A mutations) displayed reduced phosphorylation that correlated with reduced function. Replacement of both serine residues 151 and 153 with phosphomimetic aspartic acid restored Rex-2 function and locked Rex-2 in a phosphorylated active conformation. A mutant containing threonine residues at positions 151 and 153 displayed a phenotype indistinguishable from that of wild-type Rex. Furthermore, this same mutant showed increased threonine phosphorylation and decreased serine phosphorylation, providing conclusive evidence that one or both of these residues are phosphorylated in vivo. Our results provide the first direct evidence that the phosphorylation of Rex-2 is important for function. Further understanding of HTLV Rex phosphorylation will provide insight into the regulatory control of HTLV replication and ultimately the pathobiology of HTLV

The Glycogen-Bound Polyphosphate Kinase from Sulfolobus acidocaldarius Is Actually a Glycogen Synthase

Inorganic polyphosphate (polyP) is obtained by the polymerization of the terminal phosphate of ATP through the action of the enzyme polyphosphate kinase (PPK). Despite the presence of polyP in every living cell, a gene homologous to that of known PPKs is missing from the currently sequenced genomes of Eukarya, Archaea, and several bacteria. To further study the metabolism of polyP in Archaea, we followed the previously published purification procedure for a glycogen-bound protein of 57 kDa with PPK as well as glycosyl transferase (GT) activities from Sulfolobus acidocaldarius (R. Skórko, J. Osipiuk, and K. O. Stetter, J. Bacteriol. 171:5162–5164, 1989). In spite of using recently developed specific enzymatic methods to analyze polyP, we could not reproduce the reported PPK activity for the 57-kDa protein and the polyP presumed to be the product of the reaction most likely corresponded to glycogen-bound ATP under our experimental conditions. Furthermore, no PPK activity was found associated to any of the proteins bound to the glycogen-protein complex. We cloned the gene corresponding to the 57-kDa protein by using reverse genetics and functionally characterized it. The predicted product of the gene did not show similarity to any described PPK but to archaeal and bacterial glycogen synthases instead. In agreement with these results, the recombinant protein showed only GT activity. Interestingly, the GT from S. acidocaldarius was phosphorylated in vivo. In conclusion, our results convincingly demonstrate that the glycogen-protein complex of S. acidocaldarius does not contain a PPK activity and that what was previously reported as being glycogen-bound PPK is a bacterial enzyme-like thermostable glycogen synthase