Interaction of PKN with α-Actinin

Abstract

PKN is a fatty acid-and Rho-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. To identify components of the PKN-signaling pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed. Using the N-terminal region of PKN as a bait, cDNAs encoding actin cross-linking protein ␣-actinin, which lacked the N-terminal actin-binding domain, were isolated from human brain cDNA library. The responsible region for interaction between PKN and ␣-actinin was determined by in vitro binding analysis using the various truncated mutants of these proteins. The N-terminal region of PKN outside the RhoAbinding domain was sufficiently shown to associate with ␣-actinin. PKN bound to the third spectrin-like repeats of both skeletal and non-skeletal muscle type ␣-actinin. PKN also bound to the region containing EF-hand-like motifs of non-skeletal muscle type ␣-actinin in a Ca 2؉ -sensitive manner and bound to that of skeletal muscle type ␣-actinin in a Ca 2؉ -insensitive manner. ␣-Actinin was co-immunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and ␣-actinin lacking the actin-binding domain. In vitro translated full-length ␣-actinin containing the actin-binding site hardly bound to PKN, but the addition of phosphatidylinositol 4,5-bisphosphate, which is implicated in actin reorganization, stimulated the binding activity of the full-length ␣-actinin with PKN. We therefore propose that PKN is linked to the cytoskeletal network via a direct association between PKN and ␣-actinin.PKN is a serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family in the C terminus and a unique regulatory region in the N terminus (1, 2). The N-terminal region of PKN contains repeats of leucine zipper-like motif, suggesting promotion of protein-protein association through hydrophobic interactions (3). We demonstrated that Rho, a small GTP-binding protein, binds to PKN in a GTP-dependent fashion and that this binding leads to the activation of PKN, suggesting that PKN is one of the targets of Rho (4, 5). In order to identify other proteins that interact with PKN, we have used a yeast two-hybrid system with the Nterminal region of PKN as bait. One of the positive cDNA clones isolated from human brain cDNA library encoded a neurofilament L protein, a neuron-specific intermediate filament protein (6). We have demonstrated that PKN binds to and phosphorylates the head-rod domain of intermediate filament proteins such as each subunit of neurofilament and vimentin in vitro (6) and raised the possibility that PKN plays a role in the assembly of intermediate filament, one of the major components of cytoskeleton. Here we report that the two other groups of positive cDNA clones encoded ␣-actinin, a constituent of the other major component of cytoskeleton. MATERIALS AND METHODS Two-hybrid Screens and Constructs for Two-hybrid Systems-Schemes of the fusion constructs for human PKN...

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