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.

when it is degraded to lower-molecular-weight HA (LMW-HA). 3 Other biological activities of HA are also dependent on its molecular weight 4,5 : HMW-HA is anti-inflammatory and antiangiogenic, whereas LMW-HA promotes inflammatory and angiogenic reactions. 6,7 In osteoarthritis (OA), HA degradation is enhanced and so Supported by Japan Society for the Promotion of Science Grant-in-aid for Scientific Research (JSPS KAKENHI) grants 16H05454 (Y.O.), 19H03788 (Y.O.), and 18K09082 (M.I.

The role of PKN, a fatty acid-and Rho small GTPase-activated protein kinase, in cell-cycle regulation was analyzed. Microinjection of the active form of PKN into a Xenopus embryo caused cleavage arrest, whereas normal cell division proceeded in the control embryo microinjected with buffer or the inactive form of PKN. Exogenous addition of the active form of PKN delayed mitotic timing in Xenopus egg cycling extracts judging by morphology of sperm nuclei and Cdc2͞cyclin B histone H1 kinase activity. The kinase-negative form of PKN did not affect the timing, suggesting that delayed mitotic timing depends on the kinase activity of PKN. The dephosphorylation of Tyr-15 of Cdc2 was also delayed in correlation with Cdc2͞cyclin B histone H1 kinase activation in extracts containing active PKN. The Cdc25C activity for the dephosphorylation of Tyr-15 in Cdc2 was suppressed by pretreatment with the active form of PKN. Furthermore, PKN efficiently phosphorylated Cdc25C in vitro, indicating that PKN directly inhibits Cdc25C activity by phosphorylation. These results suggest that PKN plays a significant role in the control of mitotic timing by inhibition of Cdc25C. P rotein phosphorylation͞dephosphorylation reaction is a key event in the regulation of cell division. In dividing eukaryotic cells, entry into mitosis is governed by the M phase-promoting factor. This factor consists of the Cdc2 protein kinase and cyclin B and acts by phosphorylating substrates that are essential for the execution of mitotic processes (1). Before mitosis, the activity of Cdc2͞cyclin B histone H1 kinase is suppressed through inhibitory phosphorylation of Tyr-15 and Thr-14 residues of Cdc2 by the Wee1 and Myt1 kinases (2-5). At onset of mitosis, the phosphatase Cdc25C removes these inhibitory phosphate groups from Cdc2 and thereby activates Cdc2͞cyclin B histone H1 kinase. The activity of Cdc25C is strictly regulated, probably by phosphorylation, and is low during interphase and high at mitosis (6, 7). Thus, entry into mitosis is under the control of a tightly regulated network of protein kinases and phosphatases. Although upstream players such as Chk1 and Cds1 (8-10) in the tyrosine phosphorylation͞dephosphorylation of Cdc2 have been identified, it remains unclear how these enzymes are controlled during the cell cycle. There is a possibility that the unidentified protein kinase or kinases might participate in upstream regulation.PKN is a serine͞threonine protein kinase that has a catalytic domain highly homologous to protein kinase C (PKC) in the carboxyl-terminal region and a unique regulatory domain in the amino-terminal region (11-13). The amino-terminal region of PKN contains three repeats of a leucine zipper-like motif, and its kinase activity is stimulated by fatty acids such as arachidonic acid (13). We reported that PKN translocates from the cytosol to the nucleus of fibroblasts on exposure to stress such as heat shock and serum starvation (14) and that PKN is cleaved during apoptosis, presumably by caspase-3, which generates a constitutively...

Deletion of Hybid (hyaluronan-binding protein involved in hyaluronan depolymerization) results in attenuation of osteoarthritis in mice, The American