A novel protein kinase C α-dependent signal to ERK1/2 activated by αVβ3 integrin in osteoclasts and in Chinese hamster ovary (CHO) cells

Abstract

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-amino-phenoxy)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.

Fibroblast Growth Factor Receptor 2 Promotes Osteogenic Differentiation in Mesenchymal Cells via ERK1/2 and Protein Kinase C Signaling

“…Whether PKC␣ may up-regulate directly osteoblast gene expression is an interesting question. PKC␣ was found to be involved in ERK1/2 activation in osteoclasts (64). However, we found that molecular inhibition of PKC␣ but not ERK1/2 reduced osteoblast gene expression in MT FGFR2 cells, suggesting that ERK1/2 does not act downstream of PKC␣ in these cells.…”

Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+and PIP2Recognition by Its C2 Domain

“…Our observations, however, may be cell-type specific, because other groups using similar membrane dyes have concluded that PLC␦ 1 PH domain and mGAP43 accumulation in localized regions of the plasma membrane is not caused by locally increased membrane area but instead is attributed to rafts (Huang et al, 2004;Golub and Caroni, 2005). Recently, PKC␣ has been demonstrated to associate with detergent-insoluble fractions in a Ca 2ϩ -dependent manner attributed to raft binding (Rucci et al, 2005). However, the correlation between lipid rafts in cells and detergent-insoluble fractions derived from cells continues to be challenged (van Rheenen et al, 2005).…”

Activation of P2X7 receptors causes isoform-specific translocation of protein kinase C in osteoclasts

“…For example, the extracellular matrix initiates signaling by binding to integrins. PKCα in osteoclasts associates with αvβ3 integrin and activates ERK1/2, thereby regulating cell migration and bone resorption (Rucci et al, 2005). Interestingly, αvβ3 integrin has been reported to be localized on the basolateral membrane in osteoclasts (Duong et al, 2000).…”

“…The activity of conventional protein kinase isozymes (cPKCs; α, βI, βII and γ) is stimulated by diacylglycerol, Ca 2+ and phosphatidylserine; that of novel isozymes (nPKCs; δ, ε, η and θ) by diacylglycerol and phosphatidylserine; whereas atypical PKC isoforms (aPKCs; ζ and ι) are activated by phosphatidylserine, but are independent of Ca 2+ and phorbol esters. Several PKC isozymes have been identified in osteoclasts, including α, δ and ε (Teti et al, 1995), with more recent evidence also revealing the expression of β and ζ in these cells (Williams et al, 2000;Rucci et al, 2005).…”