A novel protein kinase C -dependent signal to ERK1/2 activated by _V3 integrin in osteoclasts and in Chinese hamster ovary (CHO) cells

We identified a novel protein kinase C (PKC)-dependent signal to extracellular signal-regulated kinase (ERK)1/2 in mouse osteoclasts and Chinese hamster ovary (CHO) cells, specifically activated by the _V3 integrin. It involves translocation (i.e. activation) of PKC from the cytosol to the membrane and/or the Triton X-100-insoluble subcellular fractions, with recruitment into a complex with _V3 integrin, growth factor receptor-bound protein (Grb2), focal adhesion kinase (FAK) in CHO cells and proline-rich tyrosine kinase (PYK2) in osteoclasts. Engagement of v3 integrin triggered ERK1/2 phosphorylation, but the underlying molecular mechanism was surprisingly independent of the well known Shc/Ras/Raf-1 cascade, and of phosphorylated MAP/ERK kinase (MEK)1/2, so far the only recognized direct activator of ERK1/2. In contrast, PKC was involved in ERK1/2 activation because inhibition of its activity prevented ERK1/2 phosphorylation. The tyrosine kinase c-Src also contributed to ERK1/2 activation, however, it did not interact with PKC in the same molecular complex. The _V3/PKC complex formation was fully dependent upon the intracellular calcium concentration ([Ca²⁺]_i), and the use of the intracellular Ca²⁺ chelator 1,2-bis(o-amino-phenoxy)ethane-N,N,N9,N9-tetraaceticacidtetra (acetoxy-methyl) ester (BAPTA-AM) also inhibited PKC translocation and ERK1/2 phosphorylation. Functional studies showed that _V3 integrin-activated PKC was involved in cell migration and osteoclast bone resorption, but had no effect on the ability of cells to attach to LM609, suggesting a role in events downstream of _V3 integrin engagement.