Mapping of phosphorylation sites in human MSK1 activated by a novel interaction with MRK-beta.
The desire to map reliable phosphorylation signaling network has motivated the development of high-performance techniques. Targeted biochemical studies and updated methods employing MS techniques are most used in mapping the phosphorylation sites and verifying novel interactions of kinases. Previously, we have established a novel method to efficiently facilitate more comprehensive, accurate phosphorylation site mapping of individual phosphoproteins by using combination of multiple stage MS analysis with target-decoy database search against the much smaller targeted database. In this study, by applying this method, we have identified the phosphorylation sites in human MSK1 mitogen- and stress-activated protein kinase 1, which has been proved to be a multi-phosphorylated kinase that plays key roles in various cell functions, activated by a novel interaction with MRK-beta. The results show that this method can find out not only those previously identified active sites in MSK1, but also some novel phosphorylated sites, which correlates with biochemical evidence that, besides p38 and extracellular signal-regulated kinase, MRK-beta could also activate MSK1 through direct interaction. Hence, we conclude this method is sensitive and reliable as expected and it can be further combined with automated screening and biochemical study in efficiently building up a more comprehensive phosphoprotein network.
Wang
Wei
W
State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, P. R. China.
Han
Guanghui
G
Ye
Mingliang
M
Shi
Huili
H
Zou
Hanfa
H
Huo
Keke
K
D000595:Amino Acid Sequence; D015153:Blotting, Western; D020134:Catalytic Domain; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D057166:High-Throughput Screening Assays; D006801:Humans; D020930:MAP Kinase Kinase Kinases; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods*; D010748:Phosphopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry; D020798:Two-Hybrid System Techniques
C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D010446:Peptide Fragments; D010748:Phosphopeptides; D011494:Protein Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C116919:mitogen and stress-activated protein kinase 1; D020928:Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; C413047:MAP3K20 protein, human; D010196:Pancreatic Elastase