Abstract 1535: PB1 Domain Mediated PKCz and p62 Interaction Regulates Flow-Induced ERK5 Activation
ERK5 mediates several flow-dependent endothelial cell (EC) functions including cell survival and gene expression. The upstream regulators of ERK5 stimulated by flow are poorly characterized. A protein-protein interaction domain termed PB1 was identified by proteomics as potentially involved in ERK5 activation, since MEK5 and MEKK3 (known ERK5 activators) contain PB1 domains. Therefore we hypothesized that PKCζ and p62, PB1 containing proteins prominent in EC, would regulate ERK5. PKCζ was phosphorylated and activated by shear stress (12 dyn/cm2, 2-fold increase at 10 min) in EC. Decreasing PKCζ expression by siRNA reduced significantly (50%) flow-induced ERK5 activation (measured by both kinase and luciferase assays). Inhibiting PKCζ activity with an adenoviral dominant-negative PKCζ blocked ERK5 activation. PKCζ signals downstream of MEK5 since PKCζ siRNA blocked effects of overexpressed constitutive active (CA) MEK5 on ERK5 activity. In contrast, PKCζ wild type overexpression increased CA-MEK5 activation of ERK5. These observations demonstrate a positive role for PKCζ in ERK5 regulation. Assembly of a MEKK3-MEK5-ERK5-PKCζ signalosome likely involves a scaffold protein. Because p62 is a well-known PB1 scaffold, we studied its interactions with ERK5 and PKCζ in EC. We found that p62 and ERK5 interacted basally and flow decreased the ERK5/p62 interaction. Overexpressing p62 wild type inhibited flow-induced ERK5 activity and increased EC apoptosis. In response to flow (12 dyn/cm2, 10 min), p62 associated with PKCζ. We propose that the scaffold p62 binds and inhibits ERK5 basally. Flow activates PKCζ which now recruits p62 and allows MEK5 to bind and activate ERK5. This study defines flow-mediated interactions between PB1-domain containing proteins that control ERK5 activity, and suggests that PKCζ and p62 are novel regulators of EC survival and gene expression.