Platelet sarco/endoplasmic reticulum Ca2+ATPase isoform 3b and Rap 1b: interrelation and regulation in physiopathology.
Platelet Ca2+ signalling involves intracellular Ca2+ pools, whose content is controlled by sarco/endoplasmic reticulum Ca2+ATPases (SERCAs). Among these, a key role is played by the inositol trisphosphate-sensitive Ca2+ pool, associated with the SERCA 3b isoform. We have investigated the control of this Ca2+ pool through the cAMP-dependent phosphorylation of the GTP-binding protein, Rap (Ras-proximate) 1b. We first looked for this Ca2+ pool target of regulation by studying the expression of the different SERCA and Rap 1 proteins in human platelets and various cell lines, by Western blotting and reverse transcription-PCR. Since co-expression of Rap 1b and SERCA 3b was obtained, we looked for their protein-protein interaction as a function of the cAMP-dependent phosphorylation of Rap 1b. Co-immunoprecipitations of SERCA 3b and Rap 1b proteins were found in the absence of phosphorylation, induced by the catalytic subunit of the cAMP-dependent protein kinase (csPKA). In contrast, upon pre-treatment of platelet membranes with csPKA, the SERCA 3b dissociated from the Rap 1b protein, in agreement with a role of its phosphorylated state in their interaction. Finally, we looked for adaptation of this complex in a platelet pathological model of hypertension. We investigated the expression of both proteins, as well as the cAMP-dependent phosphorylation of Rap 1b and SERCA 3b activity in platelets from control normotensive Wistar-Kyoto rats and from spontaneously hypertensive rats (SHRs). A decrease in SERCA 3b activity was associated with a decrease in Rap 1b endogenous phosphorylation in SHR platelets, consistent with a functional role in the regulation of the SERCA 3b-associated Ca2+ pool.
Lacabaratz-Porret
C
C
Institut National de la Santé et de la Recherche Médicale U 348, IFR Circulation Lariboisière, Hôpital Lariboisière, 8 rue Guy Patin, 75475 Paris Cedex 10, France.
Corvazier
E
E
Kovàcs
T
T
Bobe
R
R
Bredoux
R
R
Launay
S
S
Papp
B
B
Enouf
J
J
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D019204:GTP-Binding Proteins / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006801:Humans; D006973:Hypertension / Q000503:physiopathology; D007295:Inositol Phosphates / Q000502:physiology; D007527:Isoenzymes / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011918:Rats, Inbred SHR; D017208:Rats, Wistar
D007295:Inositol Phosphates; D007527:Isoenzymes; D012333:RNA, Messenger; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000252:Calcium-Transporting ATPases