Masaki Shimakawa

A novel 450-kDa coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was identified as a protein that interacted with the regulatory region of the protein kinase PKN, having a catalytic domain homologous to that of protein kinase C. CG-NAP contains two sets of putative RII (regulatory subunit of protein kinase A)-binding motif. Indeed, CG-NAP tightly bound to RIIalpha in HeLa cells. Furthermore, CG-NAP was coimmunoprecipitated with the catalytic subunit of protein phosphatase 2A (PP2A), when one of the B subunit of PP2A (PR130) was exogenously expressed in COS7 cells. CG-NAP also interacted with the catalytic subunit of protein phosphatase 1 in HeLa cells. Immunofluorescence analysis of HeLa cells revealed that CG-NAP was localized to centrosome throughout the cell cycle, the midbody at telophase, and the Golgi apparatus at interphase, where a certain population of PKN and RIIalpha were found to be accumulated. These data indicate that CG-NAP serves as a novel scaffolding protein that assembles several protein kinases and phosphatases on centrosome and the Golgi apparatus, where physiological events, such as cell cycle progression and intracellular membrane traffic, may be regulated by phosphorylation state of specific protein substrates.

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 α-actinin, which lacked the N-terminal actin-binding domain, were isolated from human brain cDNA library. The responsible region for interaction between PKN and α-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 α-actinin. PKN bound to the third spectrin-like repeats of both skeletal and non-skeletal muscle type α-actinin. PKN also bound to the region containing EF-hand-like motifs of non-skeletal muscle type α-actinin in a Ca2+-sensitive manner and bound to that of skeletal muscle type α-actinin in a Ca2+-insensitive manner. α-Actinin was co-immunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and α-actinin lacking the actin-binding domain. In vitro translated full-length α-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 α-actinin with PKN. We therefore propose that PKN is linked to the cytoskeletal network via a direct association between PKN and α-actinin. 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 α-actinin, which lacked the N-terminal actin-binding domain, were isolated from human brain cDNA library. The responsible region for interaction between PKN and α-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 α-actinin. PKN bound to the third spectrin-like repeats of both skeletal and non-skeletal muscle type α-actinin. PKN also bound to the region containing EF-hand-like motifs of non-skeletal muscle type α-actinin in a Ca2+-sensitive manner and bound to that of skeletal muscle type α-actinin in a Ca2+-insensitive manner. α-Actinin was co-immunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and α-actinin lacking the actin-binding domain. In vitro translated full-length α-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 α-actinin with PKN. We therefore propose that PKN is linked to the cytoskeletal network via a direct association between PKN and α-actinin.

PKN is a fatty acid-activated serine/threonine kinase that has a catalytic domain highly homologous to that of protein kinase C in the carboxyl terminus and a unique regulatory region in the amino terminus. Recently, we reported that the small GTP-binding protein Rho binds to the amino-terminal region of PKN and activates PKN in a GTP-dependent manner, and we suggested that PKN is located on the downstream of Rho in the signal transduction pathway (Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648-650; Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y. Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). To identify other components of the PKN pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed. By this screening, a clone encoding the neurofilament L protein, a subunit of neuron-specific intermediate filament, was isolated. The amino-terminal regulatory region of PKN was shown to associate with the head-rod domains of other subunits of neurofilament (neurofilament proteins M and H) as well as neurofilament L protein in yeast cells. The direct binding between PKN and each subunit of neurofilament was confirmed by using the in vitro translated amino-terminal region of PKN and glutathione S-transferase fusion protein containing the head-rod domain of each subunit of neurofilament. PKN purified from rat testis phosphorylated each subunit of the native neurofilament purified from bovine spinal cord and the bacterially synthesized head-rod domain of each subunit of neurofilament. Polymerization of neurofilament L protein in vitro was inhibited by phosphorylation of neurofilament L protein by PKN. The identification and characterization of the novel interaction with PKN may contribute toward the elucidation of mechanisms regulating the function of neurofilament.

Promoter activity of protein kinase C (PKC) gamma gene was analysed by chloramphenicol acetyltransferase (CAT) assay using extracts from the cells transfected with various fusion constructs containing the 5'-flanking region of the mouse PKC gamma gene and CAT gene. Transient expression experiments in PC12 cells revealed that the upstream region of 87 bp from the transcriptional initiation site was sufficient for promoter activity. The region containing nucleotides 87 upstream from the transcriptional initiation site was shown to silence CAT activity in Balb/c3T3 cells, in which mRNA of PKC gamma was not detected, suggesting that this region might contain a transcriptional regulatory element for the cell type-specific expression of the PKC gamma gene.