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.

The vertebrate olfactory system discriminates a wide variety of odorants by relaying coded information from olfactory sensory neurons in the olfactory epithelium to olfactory cortical areas of the brain. Recent studies have shown that the first step in odor discrimination is mediated by ϳ1000 distinct olfactory receptors, which comprise the largest family of G-protein-coupled receptors. In the present study, we used Ca 2ϩ imaging and single-cell reverse transcription-PCR techniques to identify mouse olfactory neurons responding to an odorant and subsequently to clone a receptor gene from the responsive cell. The functionally cloned receptors were expressed in heterologous systems, demonstrating that structurally related olfactory receptors recognized overlapping sets of odorants with distinct affinities and specificities. Our results provide direct evidence for the existence of a receptor code in which the identities of different odorants are specified by distinct combinations of odorant receptors that possess unique molecular receptive ranges. We further demonstrate that the receptor code for an odorant changes with odorant concentration. Finally, we show that odorant receptors in human embryonic kidney 293 cells couple to stimulatory G-proteins such as G␣olf, resulting in odorant-dependent increases in cAMP. Odor discrimination is thus determined by differences in the receptive ranges of the odorant receptors that together encode specific odorant molecules. Key words: olfactory; odorant; receptor; single-cell RT-PCR; calcium imaging; G-protein; cAMP; HEK293In the olfactory system, sensory information is processed through a series of distinct anatomical structures beginning at the olfactory epithelium in the nose, in which a variety of odorant molecules are detected, and ending at the higher cortical areas of the brain, in which a perception is constructed

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.

RAC protein kinase (RAC-PK), a serine/ threonine protein kinase containing a pleckstrin homology (PH) domain, was activated by cellular stress such as heat shock and hyperosmolarity. Wortmannin, which is known as a potent inhibitor of phosphatidylinositol 3-kinase and normally inhibits growth factor-induced activation of RAC-PK, did not suppress heat-shock induced activation of RAC-PK, indicating that this stress-induced activation of the kinase is not mediated by phosphatidylinositol 3-kinase. The PH domain was indispensable for stress-induced activation of RAC-PK. In heat-treated cells, PKC 8, a member of the protein kinase C family, was found to associate with the PH domain of RAC-PK. This PKC subspecies was phosphorylated in vitro by RAC-PK. The results suggest that RAC-PK may play a role in the cellular response to stress through its PH domain.The pleckstrin homology (PH) domain was originally defined as repeated amino acid sequences of 100 to 110 residues in pleckstrin, the major protein kinase C (PKC) substrate in platelets, and has subsequently been found in many intracellular signaling proteins and cytoskeletal proteins (1-3). Although the sequence similarity of the PH domains is low, the peptide folds of the domain from different proteins such as pleckstrin (4), ,3-spectrin (5), and dynamin (6, 7) are virtually the same, composing of seven antiparallel }3-sheets and an a-helix in the N-and C-terminals, respectively. The common structure and wide distribution among various signaling proteins suggest that the PH domain serves as a functional region involved in the intracellular signaling network. Several proteins such as the 13y subunits -of GTP-binding regulatory proteins (8-12) and the PKC subspecies (12-15) as well as phosphatidylinositol 4,5-bisphosphate (16) and inositol 1,4,5-trisphosphate (17-20) are shown to associate with the distinct regions of the PH domain, suggesting that the PH domain conveys information by interacting with various signaling molecules.RAC-protein kinase (RAC-PK, also named as PKB or Akt) was identified as a serine/threonine protein kinase (PK) having a catalytic domain closely related to both cAMPdependent PK and PKC (12,14,[21][22][23], and also as a cellular homologue of the viral oncogene v-akt (24, 25). The catalytic domain of RAC-PK is located in its C-terminal region, and the N-terminal end region contains a PH domain. Three subtypes of RAC-PK, termed a, ,B, and 'y, have been isolated (12,14,(21)(22)(23)(24)(25). The RAC-PK subtypes are shown to associate with PKC subspecies as well as the fry subunits of GTP-binding regulatory proteins in vitro (12,14,15). RAC-PK is, therefore, supposed to be involved in the cell growth regulation, as its viral version is an oncogene and RAC-PK interacts with some proteins engaged in the transduction of signals from growth factors. Recently, RAC-PK was indeed shown to be activated by platelet-derived growth factor (PDGF) in NIH 3T3, Rat-1, and MEF cells (26,27), by epidermal growth factor and basic fibroblast growth factor ...

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