Mutational analysis of the regulatory mechanism of PKN: the regulatory region of PKN contains an arachidonic acid-sensitive autoinhibitory domain.

PKN is a fatty acid- and Rho GTPase-activated protein kinase whose catalytic domain in the carboxyl terminus is homologous to those of protein kinase C (PKC) family members. The amino terminal region of PKN is suggested to function as a regulatory domain, since tryptic cleavage or the binding of Rho GTPase to this region results in protein kinase activation of PKN. The structural basis for the regulation of PKN was investigated by analyzing the activity of a series of deletion/site-directed mutants expressed in insect cells. The amino-terminally truncated form of PKN (residue 455-942) showed low basal activity similar to that of the wild-type enzyme, and was arachidonic acid-dependent. However, further deletion (residue 511-942) resulted in a marked increase in the basal activity and a decrease in the arachidonic acid dependency. A (His)(6)-tagged protein comprising residues 455-511 of PKN (designated His-Ialpha) inhibited the kinase activity of the catalytic fragment of PKN in a concentration-dependent manner in competition with substrate (K(i) = 0.6+/-0.2 microM). His-Ialpha also inhibited the activity of the catalytic fragment of PRK2, an isoform of PKN, but had no inhibitory effect on protein kinase A or protein kinase Cdelta. The IC(50) value obtained in the presence of 40 microM arachidonic acid was two orders of magnitude greater than that in the absence of the modifier. These results indicate that this protein fragment functions as a specific inhibitor of PKN and PRK2, and that arachidonic acid relieves the catalytic activity of wild-type PKN from autoinhibition by residues 455-511 of PKN. Autophosphorylation of wild-type PKN increased the protein kinase activity, however, substitution of Thr64, Ser374, or Thr531 in the regulatory region of PKN with alanine, abolished this effect. Substitution of Thr774 in the activation loop of the catalytic domain of PKN with alanine completely abolished the protein kinase activity. These results suggest that these phosphorylation sites are also important in the regulation of the PKN kinase activity. Potential differences in the mechanism of activation between the catalytic regions of PKN and PRK2 are also discussed.

Proteolytic activation of PKN by caspase-3 or related protease during apoptosis.

PKN, a fatty acid- and Rho-activated serine/threonine kinase having a catalytic domain highly homologous to protein kinase C (PKC), was cleaved at specific sites in apoptotic Jurkat and U937 cells on Fas ligation and treatment with staurosporin or etoposide, respectively. The cleavage of PKN occurred with a time course similar to that of PKCdelta, a known caspase substrate. This proteolysis was inhibited by a caspase inhibitor, acetyl-Asp-Glu-Val-Asp-aldehyde. The cleavage fragments were generated in vitro from PKN by treatment with recombinant caspase-3. Site-directed mutagenesis of specific aspartate residues prevented the appearance of these fragments. These results indicate that PKN is cleaved by caspase-3 or related protease during apoptosis. The major proteolysis took place between the amino-terminal regulatory domain and the carboxyl-terminal catalytic domain, and it generated a constitutively active kinase fragment. The cleavage of PKN may contribute to signal transduction, eventually leading to apoptosis.

PKN interacts with a paraneoplastic cerebellar degeneration-associated antigen, which is a potential transcription factor.

PKN is a fatty acid-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. PKN has been recently reported to interact with a small GTP-binding protein Rho and cytoskeletal proteins such as neurofilament and alpha-actinin. To identify the new components of the PKN-signaling pathway, the yeast two-hybrid system was employed. Using the amino-terminal regulatory domain of PKN as a bait, cDNA encoding a neural antigen PCD17, which is recognized by characteristic antibodies of patients with paraneoplastic cerebellar degeneration, was isolated from a human brain cDNA library. The interaction between PKN and PCD17 was also determined by the in vitro binding analysis. PCD17 was coimmunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and the amino-terminal region of PCD17. PCD17 was phosphorylated by PKN, and the extent of this phosphorylation was enhanced by addition of 40 microM arachidonic acid. The amino-terminal region of PCD17 could form a homodimer in vitro, and PCD17 fused to the Gal4 DNA binding domain showed the transcriptional transactivation of the chloramphenicol acetyltransferase reporter gene linked to 5 Gal4 binding sites and minimal promoter in rat C6 glioma cells. These results suggest the participation of PCD17 in gene expression and lead to a clue for elucidating the PKN signaling pathway from the cytosol to the nucleus.

Interaction of PKN with alpha-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 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.

Translocation of PKN from the cytosol to the nucleus induced by stresses.

Effects of environmental stresses on the subcellular localization of PKN were investigated in NIH 3T3, BALB/c 3T3, and Rat-1 cells. The immunofluorescence of PKN resided prominently in the cytoplasmic region in nonstressed cells. When these cells were treated at 42 degrees C, there was a time-dependent decrease of the immunofluorescence of PKN in the cytoplasmic region that correlated with an increase within the nucleus as observed by confocal microscope. After incubation at 37 degrees C following beat shock, the immunofluorescence of PKN returned to the perinuclear and cytoplasmic regions from the nucleus. The nuclear translocation of PKN by heat shock was supported by the biochemical subcellular fractionation and immunoblotting. The nuclear localization of PKN was also observed when the cells were exposed to other stresses such as sodium arsenite and serum starvation. These results raise the possibility that there is a pathway mediating stress signals from the cytosol to the nucleus through PKN.

PKN associates and phosphorylates the head-rod domain of neurofilament protein.

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.

The role of the unique motifs in the amino-terminal region of PKN on its enzymatic activity.

The yeast two-hybrid system and in vitro binding assay were carried out to characterize the interaction between the amino-terminal and carboxyl-terminal region of PKN. It was revealed that the amino-terminal region containing the regulatory domain associated with the carboxyl-terminal catalytic region. A synthetic peptide, corresponding to the amino acid residues of PKN from 39 to 53, with substitution of isoleucine46 with serine was shown to become a potent substrate for PKN, and its wild type synthetic peptide inhibited the phosphorylation by PKN. These results suggest that the amino-terminal region of PKN contains the pseudosubstrate sequence and acts as an autoinhibitory region.