Protein kinase C (PKC) isoforms, ␣, ␤I, and ␥ of cPKC subgroup, ␦ and of nPKC subgroup, and of aPKC subgroup, were tyrosine phosphorylated in COS-7 cells in response to H 2 O 2 . These isoforms isolated from the H 2 O 2 -treated cells showed enhanced enzyme activity to various extents. The enzymes, PKC ␣ and ␦, recovered from the cells were independent of lipid cofactors for their catalytic activity. Analysis of mutated molecules of PKC ␦ showed that tyrosine residues, which are conserved in the catalytic domain of the PKC family, are critical for PKC activation induced by H 2 O 2 . These results suggest that PKC isoforms can be activated through tyrosine phosphorylation in a manner unrelated to receptor-coupled hydrolysis of inositol phospholipids.

Protein kinase C ␦ (PKC ␦) is normally activated by diacylglycerol produced from receptor-mediated hydrolysis of inositol phospholipids. On stimulation of cells with H2O2, the enzyme is tyrosine phosphorylated, with a concomitant increase in enzymatic activity. This activation does not appear to accompany its translocation to membranes. In the present study, the tyrosine phosphorylation sites of PKC ␦ in the H2O2-treated cells were identified as Tyr-311, Tyr-332, and Tyr-512 by mass spectrometric analysis with the use of the precursor-scan method and by immunoblot analysis with the use of phosphorylation site-specific antibodies. Tyr-311 was the predominant modification site among them. In an in vitro study, phosphorylation at this site by Lck, a non-receptor-type tyrosine kinase, enhanced the basal enzymatic activity and elevated its maximal velocity in the presence of diacylglycerol. The mutation of Tyr-311 to phenylalanine prevented the increase in this maximal activity, but replacement of the other two tyrosine residues did not block such an effect. The results indicate that phosphorylation at Tyr-311 between the regulatory and catalytic domains is a critical step for generation of the active PKC ␦ in response to H2O2. P rotein kinase C (PKC) comprises a family of more than ten serine͞threonine protein kinases that are involved in a variety of signal transduction pathways (1). Each isoform has the regulatory and catalytic domains in the amino-and carboxylterminal halves, respectively. The isoforms are divided into three groups, cPKC, nPKC, and aPKC, because of the structural differences in their regulatory domains. The cPKC and nPKC isoforms are activated by diacylglycerol produced from receptormediated hydrolysis of inositol phospholipids and are the prime targets of tumor-promoting phorbol esters that bind to the cysteine-rich sequence, named the C1 region, in the regulatory domain. In general, a number of protein kinases are known to be controlled by phosphorylation (2), and the PKC family members have three phosphorylation motif sites mostly conserved among the family (3). One is a threonine residue in the activation loop of the catalytic domain, and the others are serine and threonine residues located in the carboxyl-terminal end region, named the turn and hydrophobic motifs, respectively.The PKC isoforms are further phosphorylated on tyrosine upon stimulation of the cells (4), and the role of tyrosine phosphorylation has been investigated for PKC ␦, a member of the nPKC group (5). That is, PKC ␦ is phosphorylated on tyrosine in v-ras-transformed keratinocytes (6) and in various cells stimulated with phorbol ester, growth factors, and hormones (7-14). However, controversial results are reported on the functional consequence of the tyrosine phosphorylation reaction induced by these membrane-coupled signaling processes. In keratinocytes, tyrosine phosphorylation reduces its catalytic activity (6, 12), whereas in other cases the modification reaction enhances the enzymatic activity (4, 7, 8) or even alters...

Protein kinase B (PKB, also named as Akt or RACprotein kinase), that is activated by cellular stress such as heat shock and hyperosmotic treatment, was revealed to be activated by oxidative stress and by chemical stressors of CdCl 2 and NaAs0 2 by measuring the activity of the enzyme immunoprecipitated from the transfected COS-7 cells. Upon stress treatment, a 30-kDa phosphoprotein was co-immunoprecipitated with PKB from the cells metabolic labeled with | 32 P]orthophosphate. The phosphoprotein was identified as Hsp27, a small heat shock protein, by immunoblot analysis and co-immunoprecipitation. The association of Hsp27 was specific to PKB as the heat shock protein was not co-immunoprecipitated with other protein kinases such as protein kinase C and PKN. When the cells were treated with H 2 0 2 , PKB was activated gradually and the association of Hsp27 with PKB increased concurrently with the enhancement of PKB activity. In heat-shocked cells, activation of PKB and the association of Hsp27 were detected immediately after the treatment, and the association of the heat shock protein decreased while PKB kept stimulated activity when the cells were further incubated at 37°C. These results suggest that Hsp27 is involved in the activation process of PKB in the signal transduction pathway of various forms of stress.

No abstract