Correlated expression of the 97 kDa sarcoendoplasmic reticulum Ca2+-ATPase and Rap1B in platelets and various cell lines

Abstract

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.

Endoplasmic reticulum Ca2+-ATPase pump is up-regulated in calcium-transporting dental enamel cells: a non-housekeeping role for SERCA2b

“…Recognition of SERCA2b as a generic housekeeper followed evidence of universal cellular distribution, and constitutive expression during a variety of developmental and pathophysiological situations [19,20,28]. The production of SERCA2-knockout mice established that SERCA2b is essential for life, consistent with a fundamental housekeeping function [45].…”

“…The acid-stable SERCA phosphoenzyme intermediate was assayed essentially as described previously [28]. Control experiments verified that the observed $#P-phosphoenzyme bands (most intense at 90 kDa, but extending variably from 115-70 kDa) exhibited characteristic properties including : rapid turnover during a cold-ATP chase ; sensitivity to EGTA, vanadate and lanthanum ; exclusive localization in the particulate fraction (results not shown).…”

cAMP-Binding Protein Epac Induces Cardiomyocyte Hypertrophy

“…27 Interestingly, the small GTPase Rap1 which is an effector of Epac is suspected to play a role in cAMP-induced [Ca 2ϩ ] i increase via SERCA3b in megakaryocytes. 28,29 Therefore, one could imagine in cardiac myocytes, that Epac might interact with Ca 2ϩ release channels. Such hypothesis is currently undergoing investigation.…”

All three splice variants of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene are translated to proteins: a study of their co-expression in platelets and lymphoid cells

“…In earlier studies we have demonstrated that the KATO III gastric cancer cell line expresses the SERCA2b isoform, but not the SERCA3a protein [19][20][21]. Therefore, microsomal membranes from KATO III cells (25 µg\lane) were analysed for immunostaining by the ChS3a, ChS3b and ChS3c antibodies ( Figure 2B, lanes 1, 3 and 5, respectively).…”

“…In human non-muscle tissues the housekeeping SERCA2b [15,16] and a SERCA3 isoform [17,18] (SERCA3a according to the recent nomenclature) have been identified. The presence of a SERCA protein recognized by PL\IM 430, a monoclonal anti-SERCA antibody raised against highly purified platelet intracellular membranes, has also been demonstrated [19,20] ; however, the identity of this SERCA form has long been debated [21][22][23]. While two alternatively spliced variants have long been known for both SERCA1 and SERCA2 [14][15][16]24,25], alternative splicing of the human SERCA3 pre-mRNA has only recently been reported by three independent groups [26][27][28].…”