Correlated expression of the 97 kDa sarcoendoplasmic reticulum Ca(2+)-ATPase and Rap1B in platelets and various cell lines.
Evidence has accumulated that cyclic AMP (cAMP)-induced phosphorylation of a Ras-related protein (Rap1) regulates platelet Ca2+ transport. As this transport was recently found to be controlled by two isoforms of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA), the 100 kDa SERCA2b and the newly identified 97 kDa SERCA, we attempted to establish which isoform is involved in this regulation. For this purpose, we studied the expression and regulation of both the SERCA and Rap1 isoforms in platelets, haemopoietic cells and various cancer cell lines. SERCA2b was shown to be equally expressed in all the cell lines tested, as determined by detection of its phosphoenzyme formation and by Western blotting using an isoform-specific antibody. In contrast, the expression of the 97 kDa SERCA, studied by the same methods, varied from total absence in the cancer cells to high levels in the megakaryocytic cell lines. With regard to the potential regulatory Rap1 proteins, Western blotting showed different expression of total Rap1 isoforms among the cell lineages, thus ruling out any possible relationship between Rap1 and SERCA2b. However, the expression of Rap1 proteins correlated with that of the 97 kDa SERCA isoform. More refined analysis of the rap1A and rap1B isoforms by reverse transcription PCR and by determining cAMP-induced phosphorylation of Rap1B, i.e. its functional mechanism, confirmed the correlation between Rap1B and the 97 kDa SERCA expression. This relationship was also established by the concerted up-regulation of these two proteins demonstrated in the pathological model of platelets from hypertensive rats. It is concluded that the expressions of 97 KDa SERCA and Rap1B are related, suggesting that regulation of the platelet Ca(2+)-ATPase system by cAMP-induced phosphorylation of Rap1B specifically involves the 97 kDa SERCA.
Magnier
C
C
INSERM U348, Hôpital Lariboisière, Paris, France.
Bredoux
R
R
Kovacs
T
T
Quarck
R
R
Papp
B
B
Corvazier
E
E
de Gunzburg
J
J
Enouf
J
J
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D020668:rap GTP-Binding Proteins
D007527:Isoenzymes; D012333:RNA, Messenger; D000242:Cyclic AMP; D011494:Protein Kinases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D000252:Calcium-Transporting ATPases