PKN, a fatty acid-and Rho-activated serine͞ 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 PKC␦, 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.
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.
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...
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
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°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°C following heat 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 is a fatty acid-activated serine/threonine protein kinase that has a catalytic domain highly homologous to that of protein kinase C in the carboxyl terminus and contains a unique regulatory region in the amino terminus (1-3). Recently, we demonstrated that Rho, a small GTPase protein, binds to PKN in a GTP-dependent fashion, and that this binding leads to the activation of PKN (4,5), suggesting that PKN is one of the targets of Rho. Rho is implicated in the organization of cytoskeleton in response to growth factors such as the formation of stress fibers and focal adhesions. Rho regulates cytoskeletal rearrangements, such as cell morphology (6), platelet aggregation (7,8), cell motility (9), and cytokinesis (10, 11). Rho also has roles in signaling to the nucleus and the regulation of transcriptional activation (12), cell-cycle progression (13), and cell transformation (14). Thus, the targets of the signaling pathway of Rho seem to be located within several cellular compartments. Signal transduction therefore requires the localization of Rho and Rho-regulated signaling molecules in each subcellular compartment that contains physiologically relevant roles mediated by Rho. We have reported that PKN associates and phosphorylates the intermediate filament proteins in vitro, indicating that the regulation of the cytoskeletal components was one of the possible functions of PKN (15).Recently, increasing evidence indicates that there is overlapping of the growth factor-and stress-signaling pathways. Rac and Cdc42Hs, other members of the Rho family small GTPases, are activated not only by growth factors but by stresses such as proinflammatory cytokines and ultraviolet radiation, and contribute to activation of stress-activated mitogen-activating protein kinases (16)(17)(18). However, little is known about the Rho-mediated signaling pathways of stresses. In this report, we investigate the effects of various stresses on the subcellular localization of PKN in culture cells and present a possibility that PKN, one of the targets of Rh...
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