PKN delays mitotic timing by inhibition of Cdc25C: possible involvement of PKN in the regulation of cell division.

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 Cdc2/cyclin 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 Cdc2/cyclin 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.

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.

Onset of depressive episodes in a woman with seasonal affective disorder of "spring type" coincident with atmospheric temperature, but not with sunshine duration.

This is a report on the clinical symptoms and the longitudinal course in a woman with seasonal affective disorder of "spring type." The woman experienced 10 atypical depressive episodes between the ages of 44 and 55. Nine of the 10 depressive episodes were confirmed by the medical chart, and were classified into three categories: two episodes with a psychological stressor, six episodes with the onset in February or March, and an episode starting in August. The onset of the six episodes did coincide with the time the atmospheric temperature rose to 8 degrees Centigrade in spring, but did not relate with the sunshine duration. These findings suggest that the change in atmospheric temperature played an important role in triggering off her depressive episodes.