Evaluation of the expression and intracellular localization of a 44-kDa calmodulin binding protein during exponential growth and quiescence (G0)

Abstract

We have previously demonstrated that changes in calmodulin (CaM) levels are associated with G/S transition of the cell cycle and entry into and release from quiescence (G0). CaM mediates its regulation through the specific interaction with different intracellular proteins called calmodulin binding proteins (CaMBPs). This study was designed to evaluate the expression of the CaMBPs during the cell cycle. Mouse C127 cells were synchronized in quiescence (G0) by serum deprivation. Analysis of the CaMBPs by the125I-labeled CaM ([125I]CaM) overlay procedure on one- and two-dimensional gels revealed many proteins that bind to CaM at any given time during the cell cycle. However, specific expression of a 44-kiloDalton CaMBP (44CaMBP) was observed. As cells entered quiescence (G0) phase, there was a decrease in the CaM binding to the 44CaMBP. During release into the cell cycle from G0phase, 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 during S phase and decreased as the cells progressed into G2/M. Antibody directed against the 44CaMBP was produced in rabbit. Quantitation of the 44CaMBP by Western blot analysis revealed a similar pattern to that observed by die [125I]CaM overlay procedure during the course of G0entry and release. The anti-44CaMBP antibody was used to evaluate the intracellular localization of the 44CaMBP by indirect immunofluorescence. A distinctive punctate nuclear staining, Mwas observed. This punctate nuclear staining, observed in all cells during exponential growth, disappeared as the cells entered G0. The nuclear staining remained absent in cells released from G0until the cells approached and entered the S phase, at which time the punctate nuclear staining reappeared. This staining pattern was then maintained through G2/M progression. Following M phase and entry into G1phase, the punctate nuclear staining was observed in all G1cells. Similar analysis for cells synchronized at the G1/S boundary by the double thymidine block procedure revealed that the punctate nuclear staining was present in all cells throughout the entire course of the cell cycle. The immunofluorescence staining pattern for the 44CaMBP was sensitive to the anti-CaM drug W13 at a dose that is known to reversibly block cells at G1/S. No 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. The intracellular localization, the changes in abundance, and CaM binding are consistent with a potential role for the 44CaMBP in the CaM mediated regulation of cell proliferation. Taken together, these data would suggest that the nuclear localization of the 44CaMBP is a marker for cells that are actively undergoing cell division and that abolition of this localization is associated with the cessation of this commitment.Key words: calmodulin, calmodulin binding proteins, cell cycle, two-dimensional125-labelled CaM overlay, immunofluorescence localization.