EVALUATION OF THE EXPRESSION AND INTRACELLULAR-LOCALIZATION OF A 44-KDA CALMODULIN-BINDING PROTEIN DURING EXPONENTIAL-GROWTH AND QUIESCENCE(G(0))

We have previously demonstrated that changes in calmodulin (CaM) level s are associated with G(1)/S transition of the cell cycle and entry in to and release from quiescence (G(0)). CaM mediates its regulation thr ough the specific interaction with different intracellular proteins ca lled calmodulin binding proteins (CaMBPs). This study was designed to evaluate the expression of the CaMBPs during the cell cycle. Mouse C12 7 cells were synchronized in quiescence (G(0)) by serum deprivation. A nalysis of the CaMBPs by the I-125-labeled CaM ([I-125]CaM) overlay pr ocedure on one- and two-dimensional gels revealed many proteins that b ind to CaM at any given time during the cell cycle. However, specific expression of a 44-kiloDalton CaMBP (44CaMBP) was observed. As cells e ntered quiescence (G(0)) phase, there was a decrease in the CaM bindin g to the 44CaMBP. During release into the cell cycle from G(0) phase, the binding to CaM was maintained at the low level, but reappeared as the cells entered S phase. CaM binding to the 44CaMBP was intense duri ng S phase and decreased as the cells progressed into G(2)/M. Antibody directed against the 44CaMBP was produced in rabbit. Quantitation of the 44CaMBP by Western blot analysis revealed a similar pattern to tha t observed by the [I-125]CaM overlay procedure during the course of G( 0) entry and release. The anti-44CaMBP antibody was used to evaluate t he intracellular localization of the 44CaMBP by indirect immunofluores cence. A distinctive punctate nuclear staining, Mwas observed. This pu nctate nuclear staining, observed in all cells during exponential grow th, disappeared as the cells entered G(0). The nuclear staining remain ed absent in cells released from G(0) until the cells approached and e ntered the S phase, at which time the punctate nuclear staining reappe ared. This staining pattern was then maintained through G(2)/M progres sion. Following M phase and entry into G(1) phase, the punctate nuclea r staining was observed in all G(1) cells. Similar analysis for cells synchronized at the G(1)/S boundary by the double thymidine block proc edure revealed that the punctate nuclear staining was present in all c ells throughout the entire course of the cell cycle. The immunofluores cence staining pattern for the 44CaMBP was sensitive to the anti-CaM d rug W13 at a dose that is known to reversibly block cells at G(1)/S. N o effect was observed by the inactive analog W12. The punctate nuclear staining of the 44CaMBP would appear to be present during all phases of the cell cycle when cells are committed to be in the cell cycle. Th e intracellular localization, the changes in abundance, and CaM bindin g are consistent with a potential role for the 44CaMBP in the CaM medi ated regulation of cell proliferation. Taken together, these data woul d suggest that the nuclear localization of the 44CaMBP is a marker for cells that are actively undergoing eel division and that abolition of this localization is associated with the cessation of this commitment .