Differential regulation of threonine and tyrosine phosphorylations on protein kinase Cdelta by G-protein-mediated pathways in platelets.
Phosphorylation of activation loop threonine (Thr(505)) and regulatory domain tyrosine (Tyr(311)) residues are key regulators of PKC (protein kinase C) delta function in platelets. In the present study, we show that G(q) and G(12/13) pathways regulate the Thr(505) and Tyr(311) phosphorylation on PKCdelta in an interdependent manner. DiC8 (1,2-dioctanoylglycerol), a synthetic analogue of DAG (diacylglycerol), caused Thr(505), but not Tyr(311), phosphorylation on PKCdelta, whereas selective activation of G(12/13) pathways by the YFLLRNP peptide failed to cause phosphorylation of either residue. However, simultaneous activation by DiC8 and YFLLRNP resulted in Thr(505) and Tyr(311) phosphorylation on PKCdelta. In addition, we found that the activation of SFKs (Src family tyrosine kinases) is essential for G(12/13)-mediated Tyr(311) phosphorylation of PKCdelta. These results were confirmed using G(q)-deficient mouse platelets. Finally, we investigated whether Thr(505) phosphorylation is required for Tyr(311) phosphorylation. A T505A PKCdelta mutant failed to be phosphorylated at Tyr(311), even upon stimulation of both G(q) and G(12/13) pathways. We conclude that (i) PKCdelta binding to DAG, downstream of G(q) pathways, and its translocation results in Thr(505) phosphorylation, (ii) G(12/13) pathways activate SFKs required for the phosphorylation of Tyr(311) on Thr(505)-phosphorylated PKCdelta, and (iii) Thr(505) phosphorylation is a prerequisite for Tyr(311) phosphorylation on PKCdelta.
Murugappan
Swaminathan
S
Department of Physiology, Temple University School of Medicine, 3420 N Broad Street, Philadelphia, PA 19140, USA.
Chari
Ramya
R
Palli
Vasu M
VM
Jin
Jianguo
J
Kunapuli
Satya P
SP
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D004075:Diglycerides / Q000378:metabolism / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation / Q000187:drug effects; D051745:Protein Kinase C-delta / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D013912:Threonine / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
C011439:1,2-diacylglycerol; D004075:Diglycerides; C044295:1,2-dioctanoylglycerol; D013912:Threonine; D014443:Tyrosine; D051745:Protein Kinase C-delta; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11