title: Interaction of PKN with alpha-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 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.
authors.affiliation: Radioisotope Research Center, Kobe University, Kobe 657, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Mukai
authors.forename: M
authors.initials: M
authors.lastname: Toshimori
authors.forename: H
authors.initials: H
authors.lastname: Shibata
authors.forename: H
authors.initials: H
authors.lastname: Takanaga
authors.forename: M
authors.initials: M
authors.lastname: Kitagawa
authors.forename: M
authors.initials: M
authors.lastname: Miyahara
authors.forename: M
authors.initials: M
authors.lastname: Shimakawa
authors.forename: Y
authors.initials: Y
authors.lastname: Ono
mesh_terms: D000185:Actinin / Q000378:metabolism*; D001665:Binding Sites; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D006801:Humans; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D000185:Actinin; C061570:protein kinase N; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C