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Phosphorylation of MRP14, an S100 protein expressed during monocytic differentiation, modulates Ca(2+)-dependent
C van den Bos1, J Roth, H G Koch
1Institute of Experimental Dermatology, University of Muenster, Germany.
Abstract:
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.
Insights
Myeloid-related proteins (MRP8 and MRP14) form complexes that bind calcium and associate with cell structures. Phosphorylated MRP14 isoforms show distinct calcium binding and localization, suggesting a regulatory role in MRP8/MRP14 complexes.
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Biochemistry
Background:
- Myeloid-related proteins (MRP8 and MRP14) are calcium-binding proteins found in myelomonocytic cells.
- These proteins form complexes that interact with cellular membranes and intermediate filaments in a calcium-dependent manner.
- MRP14 exists in multiple isoforms, including MRP14' (lacking the first four amino acids) and phosphorylated forms.
Purpose of the Study:
- To investigate the translation, phosphorylation, and functional differences of MRP14 isoforms.
- To determine the role of calcium in the localization and function of MRP8/MRP14 complexes.
- To elucidate the regulatory mechanisms within MRP8/MRP14 complexes.
Main Methods:
- Metabolic labeling of monocytes with [35S]methionine to study protein translation.
- PCR analysis to investigate mRNA species.
- Two-dimensional electrophoresis, Western blotting, and autoradiography of [32P]orthophosphate-labeled proteins.
- Differential centrifugation to analyze subcellular localization.
- Isoelectric focusing and 45Ca2+ overlay assays to assess calcium binding.
Main Results:
- MRP14 and MRP14' are translated simultaneously from a single mRNA species, indicating alternative translation.
- Phosphorylated MRP14 isoforms are the primary sites of phosphate incorporation in monocytes.
- Distinct isoform patterns are observed in different subcellular locations.
- Elevated calcium levels promote the translocation of phosphorylated MRP14 isoforms to membranes and cytoskeleton.
- Phosphorylated MRP14 isoforms exhibit increased calcium binding compared to non-phosphorylated forms.
Conclusions:
- MRP14' formation results from alternative translation of a single mRNA.
- Phosphorylation significantly alters the calcium-binding properties and subcellular localization of MRP14 isoforms.
- These functional differences suggest that phosphorylated MRP14 acts as a regulatory subunit within MRP8/MRP14 complexes.