A novel protein kinase C alpha-dependent signal to ERK1/2 activated by alphaVbeta3 integrin in osteoclasts and in chinese hamster ovary (CHO) cells

We identified a novel protein kinase C (PKC)α-dependent signal to extracellular signal-regulated kinase (ERK)1/2 in mouse osteoclasts and Chinese hamster ovary (CHO) cells, specifically activated by the αVβ3 integrin. It involves translocation (i.e. activation) of PKCα from the cytosol to the membrane and/or the Triton X-100-insoluble subcellular fractions, with recruitment into a complex with αVβ3 integrin, growth factor receptor-bound protein (Grb2), focal adhesion kinase (FAK) in CHO cells and proline-rich tyrosine kinase (PYK2) in osteoclasts. Engagement of αvβ3 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, PKCα 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 PKCα in the same molecular complex. The αVβ3/PKCα complex formation was fully dependent upon the intracellular calcium concentration ([Ca2+]i), and the use of the intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraaceticacidtetra (acetoxymethyl) ester (BAPTA-AM) also inhibited PKCα translocation and ERK1/2 phosphorylation. Functional studies showed that αVβ3 integrin-activated PKCα 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 αVβ3 integrin engagement.