Rapid ubiquitination of Syk following GPVI activation in platelets.
Spleen tyrosine kinase (Syk) activation is a key intermediate step in the activation of platelets by the physiologic agonist collagen. We have found that Syk is rapidly ubiquitinated upon activation of platelets by collagen, collagen-related peptide (CRP), and convulxin. The Src family kinase inhibitors prevented Syk phosphorylation and its ubiquitination, indicating that the process is downstream of Src kinases. The ubiquitination of Syk did not cause degradation of the protein as evidenced by the lack of effect of proteasomal and lysosomal inhibitors. We separated ubiquitinated Syk from its nonubiquitinated counterpart and used an in vitro kinase assay to compare their activities. We found that the ubiquitinated Syk appeared to be about 5-fold more active. Using a phosphospecific antibody to Syk (Tyr525/Tyr526) that measures activated Syk, we found that most (60%-75%) of the active Syk is in the ubiquitinated fraction. This result explains the apparent high specific activity of ubiquitinated Syk. In c-Cbl-deficient mice, Syk is not ubiquitinated, implicating c-Cbl as the E3 ligase involved in Syk ubiquitination. Furthermore, Syk is not dephosphorylated in these mice. We propose that c-Cbl plays a regulatory role in glycoprotein VI (GPVI)/Fc receptor gamma (FcRgamma)-chain-dependent platelet activation through its interaction with Syk.
Dangelmaier
Carol A
CA
Department of Pharmacology, Temple University School of Medicine, 3420 N Broad St, Philadelphia, PA 19140, USA.
Quinter
Patricia G
PG
Jin
Jianguo
J
Tsygankov
Alexander Y
AY
Kunapuli
Satya P
SP
Daniel
James L
JL
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D003435:Crotalid Venoms / Q000494:pharmacology; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D037181:Lectins, C-Type; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D000072377:Syk Kinase; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism
D003435:Crotalid Venoms; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D037181:Lectins, C-Type; D010980:Platelet Membrane Glycoproteins; D061988:Proteasome Inhibitors; D011518:Proto-Oncogene Proteins; D014404:Tubulin; D025801:Ubiquitin; C098341:platelet membrane glycoprotein VI; D019000:Phosphotyrosine; C028890:convulxin; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D046988:Proteasome Endopeptidase Complex; C493849:Cbl protein, mouse