CORRELATED EXPRESSION OF THE 97 KDA SARCOENDOPLASMIC RETICULUM CA2-ATPASE AND RAP1B IN PLATELETS AND VARIOUS CELL-LINES()

Evidence has accumulated that cyclic AMP (cAMP)-induced phosphorylatio n of a Ras-related protein (Rap1) regulates platelet Ca2+ transport. A s this transport was recently found to be controlled by two isoforms o f sarcoendoplasmic reticulum Ca2+ 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 t he expression and regulation of both the SERCA and Rap1 isoforms in pl atelets, haemopoietic cells and various cancer cell lines. SERCA2b was shown to be equally expressed in all the cell lines tested, as determ ined by detection of its phosphoenzyme formation and by Western blotti ng using an isoform-specific antibody.,In contrast,he expression of th e 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 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 ra p1A 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 expressi on. This relationship was also established by the concerted up-regulat ion of these two proteins demonstrated in the pathological model of pl atelets from hypertensive rats. It is concluded that the expressions o f 97 kDa SERCA and Rap1B are related, suggesting that regulation of th e platelet Ca2+-ATPase system by cAMP-induced phosphorylation of Rap1B specifically involves the 97 kDa SERCA.