Phosphorylation of MRP14, an S100 protein expressed during monocytic differentiation, modulates Ca(2+)-dependent translocation from cytoplasm to membranes and cytoskeleton.

MRP8 and MRP14 are two Ca(2+)-binding proteins expressed in myelomonocytic cells. Complexes of MRP8 and MRP14 colocalize with membranes and intermediate filaments in a Ca(2+)-dependent manner. MRP14, unlike MRP8, exists in two isoforms, the smaller of which (MRP14') has been shown to lack the first four amino acids; both MRP14 and MRP14' are also present as phosphorylated forms. As shown in the present work by metabolic labeling of monocytes with [35S]methionine, MRP14 and MRP14' are translated simultaneously. By PCR analysis we found no evidence for the presence of different mRNA species. Since MRP14 is encoded by a single copy gene, our data indicate that MRP14' formation is due to alternative translation of a single mRNA species. Two-dimensional electrophoresis of [32P]orthophosphate-labeled monocyte proteins followed by Western blotting and autoradiography revealed that the two phosphorylated MRP14 isoforms incorporated the bulk of the radioactivity found in monocytic proteins. Using differential centrifugation we demonstrated the presence of distinct isoform patterns in different subcellular locations. Further, in response to elevated Ca2+ concentrations we observed a preferential translocation of phosphorylated MRP14 isoforms from the cytosol toward membranes and the cytoskeleton. This might be caused by altered calcium binding, and indeed, using isoelectric focusing and 45Ca2+ overlay the MRP14 band containing phosphorylated MRP14 revealed increased Ca2+ binding compared with bands containing other MRP14 isoforms. This represents the first evidence for functional differences in phosphorylated MRP14 isoforms compared with nonphosphorylated MRP14 isoforms. These functional differences suggest that MRP14 represents the regulatory subunit of MRP8/MRP14 complexes.