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 alpha-actinin, which lacked the N-terminal actin-binding domain, were isolated from human brain cDNA library. The responsible region for interaction between PKN and alpha-actinin was determined by in vitro binding analysis using the various truncated mutants of these proteins. The N-terminal region of PKN outside the RhoA-binding domain was sufficiently shown to associate with alpha-actinin. PKN bound to the third spectrin-like repeats of both skeletal and non-skeletal muscle type alpha-actinin. PKN also bound to the region containing EF-hand-like motifs of non-skeletal muscle type alpha-actinin in a Ca2+-sensitive manner and bound to that of skeletal muscle type alpha-actinin in a Ca2+-insensitive manner. alpha-Actinin was co-immunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and alpha-actinin lacking the actin-binding domain. In vitro translated full-length alpha-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 alpha-actinin with PKN. We therefore propose that PKN is linked to the cytoskeletal network via a direct association between PKN and alpha-actinin.
Highlights
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]
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
PKN Interacts with ␣-Actinin in the Yeast Two-hybrid System—We screened a million yeast colonies transformed with both human brain cDNA library fused to Gal4 transcriptional activation domain and a bait construct encoding PKNN1 fused to Gal4 DNA binding domain
Summary
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]. 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. We report that the two other groups of positive cDNA clones encoded ␣-actinin, a constituent of the other major component of cytoskeleton
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