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...

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in variety of receptor-stimulated cell responses. Receptors with intrinsic or associated tyrosine kinase activity recruit heterodimeric PI 3-kinases consisting of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85). We separated a PI 3-kinase that could be stimulated by the ␤␥ subunits of G protein (G␤␥) from rat liver. The G␤␥-sensitive PI 3-kinase appeared to be a heterodimer consisting of p110␤ and p85 (or their related subunits). The stimulation by G␤␥ was inhibited by the GDP-bound ␣ subunit of the inhibitory GTP-binding protein. Moreover, the stimulatory action of G␤␥ was markedly enhanced by the simultaneous addition of a phosphotyrosyl peptide synthesized according to the amino acid sequence of the insulin receptor substrate-1. Such enzymic properties could be observed with a recombinant p110␤/p85␣ expressed in COS-7 cells with their cDNAs. In contrast, another heterodimeric PI 3-kinase consisting of p110␣ and p85 in the same rat liver, together with a recombinant p110␣/p85␣, was not activated by G␤␥, although their activities were stimulated by the phosphotyrosyl peptide. These results indicate that p110␤/ p85 PI 3-kinase may be regulated in a cooperative manner by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating GTP-binding proteins.Phosphoinositide 3-kinase (PI 3-kinase) 1 is a key signaling enzyme implicated in the regulation of a broad array of biological responses including receptor-stimulated mitogenesis, oxidative burst, membrane ruffling, and glucose uptake (1, 2). The activation of PI 3-kinase results in an increase in cellular levels of D-3 phosphorylated phosphoinositides, such as PtdIns(3)P, PtdIns(3,4)P 2 , and PtdIns(3,4,5)P 3 . These products, however, do not serve as the substrates of phospholipase C (3) and thus have been proposed to act as second messengers. In this regard, recent studies have indicated that PtdIns(3,4)P 2 can directly activate certain protein kinase C and Akt (4, 5) and PtdIns(3,4,5)P 3 is capable of binding to the Pleckstrin homology domain of guanine nucleotide exchange factor of the small GTP-binding protein ARF1 (6 -8).At least two types of PI 3-kinase, in terms of mode of the activation, have been described in mammalian cells (2). One is stimulated by membrane-bound receptors activating tyrosine kinase, whereas the other is under the direct control of the heterotrimeric GTP-binding proteins. The well known former type has been structurally characterized as a heterodimer consisting of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85); the regulatory subunit contains one SH3 and two SH2 domains. Stimulation of tyrosine kinase receptors by extracellular signals phosphorylates specific tyrosine residues located in the YMXM motifs of their own receptors or adaptor molecules, such as insulin receptor substrate-1. These phosphorylated proteins bind to the SH2 domains of p85 and stimulate the...

Protein kinase C (PKC)delta was the first new/novel PKC isoform to be identified by the screening of mammalian cDNA libraries, based on the structural homology of its nucleotide sequences with those of classical/conventional PKC isoforms. PKC delta is expressed ubiquitously among cells and tissues. It is activated by diacylglycerol produced by receptor-mediated hydrolysis of membrane inositol phospholipids as well as by tumor-promoting phorbol ester through the binding of these compounds to the C1 region in its regulatory domain. It is also cleaved by caspase to generate a catalytically active fragment, and it is converted to an active form without proteolysis through the tyrosine phosphorylation reaction. Various lines of evidence indicate that PKC delta activated in distinct ways plays critical roles in cellular functions such as the control of growth, differentiation, and apoptosis. This article briefly summarizes the regulatory mechanisms of PKC delta activity and its functions in cell signaling.

Protein kinase C (PKC), a Ca2؉ /phospholipid-dependent protein kinase, is known as a key enzyme in various cellular responses, including apoptosis. However, the functional role of PKC in apoptosis has not been clarified. In this study, we focused on the involvement of PKC␦ in ceramide-induced apoptosis in HeLa cells and examined the importance of spatiotemporal activation of the specific PKC subtype in apoptotic events. Ceramide-induced apoptosis was inhibited by the PKC␦-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKC␦ expression using small interfering RNA. Ceramide induced the translocation of PKC␦ to the Golgi complex and the concomitant activation of PKC␦ via phosphorylation of Tyr 311 and Tyr 332 in the hinge region of the enzyme. Unphosphorylatable PKC␦ (mutants Y311F and Y332F) could translocate to the Golgi complex in response to ceramide, suggesting that tyrosine phosphorylation is not necessary for translocation. However, ceramide failed to activate PKC␦ lacking the C1B domain, which did not translocate to the Golgi complex, but could be activated by tyrosine phosphorylation. These findings suggest that ceramide translocates PKC␦ to the Golgi complex and that PKC␦ is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKC␦ by small interfering RNA to study the significance of phosphorylation of Tyr 311 and Tyr 332 in PKC␦ for ceramide-induced apoptosis and found that phosphorylation of Tyr 311 and Tyr 332 is indispensable for ceramide-induced apoptosis. We demonstrate here that the targeting mechanism of PKC␦, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event.

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The physico-chemical processes supporting life’s purposeful movement remain essentially unknown. Self-propelling chiral droplets offer a minimalistic model of swimming cells and, in surfactant-rich water, droplets of chiral nematic liquid crystals follow the threads of a screw. We demonstrate that the geometry of their trajectory is determined by both the number of turns in, and the handedness of, their spiral organization. Using molecular motors as photo-invertible chiral dopants allows converting between right-handed and left-handed trajectories dynamically, and droplets subjected to such an inversion reorient in a direction that is also encoded by the number of spiral turns. This motile behavior stems from dynamic transmission of chirality, from the artificial molecular motors to the liquid crystal in confinement and eventually to the helical trajectory, in analogy with the chirality-operated motion and reorientation of swimming cells and unicellular organisms.

Protein kinase B (PKB) is a downstream target of phosphatidylinositol (PI) 3-kinase in the signaling pathway of growth factors, and is activated by cellular stress such as H 2 O 2 and heat shock. To study the mechanism of the stress-induced activation of PKB, PI 3-kinase products were measured in stress-stimulated cells. Both PI 3,4-bisphosphate and PI 3,4,5-trisphosphate increased in H 2 O 2 -treated cells, and the elevation of these phospholipids and activation of PKB were concurrently blocked by wortmannin, a potent inhibitor of PI 3-kinase. In heat-shocked cells, the level of PI 3,4-bisphosphate did not change while that of PI 3,4,5-trisphosphate increased slightly, and an association between PKB molecules was observed. Two active PKB fractions, presumably monomeric and oligomeric forms, were resolved from heat-shocked cells by gel filtration column chromatography. Activation of the former was suppressed by pretreatment with wortmannin, whereas the generation and activation of the latter were not blocked by the PI 3-kinase inhibitor. Only the monomeric form, but not the oligomeric form, was recovered from H 2 O 2 -treated cells, and its activation was prevented by wortmannin. These results indicate that PKB is activated by two distinct mechanisms that are dependent and independent of PI 3-kinase in stress-stimulated cells.Key words: heat shock, hydrogen peroxide (H 2 O 2 ), phosphatidylinositol 3-kinase, pleckstrin homology domain, protein kinase B.Protein kinase B (PKB, also named Akt and RAC-protein phosphatidylinositol (PI) 3-kinase in the growth factor kinase) is a serine/threonine protein kinase with a plecksignaling pathway {3-6). Namely, PKB was first reported strin homology (PH) domain in its arnino-tenninal region to be activated by direct interaction of PI 3,4-bisphosphate and a catalytic domain in its carboxyl-terminal region (1, (PI 3,4-P 2 ) with its PH domain (7-9), and then the phos-2). This protein kinase was identified as an enzyme with a phorylation of PKB was shown to be indispensable for its catalytic domain closely related to both . In PKBa, Thr 308 in the activation loop protein kinase and protein kinase C, and also as a cellular of its catalytic domain and Ser 473 in the carboxyl-terminal counterpart of a rodent viral oncogene v-akt. Thus far, end region were identified as the phosphorylation sites three mammalian PKB genes, a, fi, and y, have been (12). Later, PDK1 (3-phosphoinositide-dependent protein isolated. Studies on physiological roles of PKB have revealkinase) and related enzymes were isolated that catalyze the ed that this protein kinase is a downstream target of phosphorylation of Thr 308 of PKB a-in the presence of PI 3, 4,5-trisphosphate (PI 3,4,5-P 3 ) and PI 3,4-P 2 (13-16). 'This study was supported in part by research grants from the Thus, the direct association of the PI 3 -kinase products with Scientific Research Funds of the Ministry of Education, Science, the PH domain and phosphorylation by upstream protein Sports and Culture of Japan, the Suntory Institute for Bi...

Collective migration of epithelial cells plays crucial roles in various biological processes such as cancer invasion. In migrating epithelial sheets, leader cells form lamellipodia to advance, and follower cells also form similar motile apparatus at cell–cell boundaries, which are called cryptic lamellipodia (c-lamellipodia). Using adenocarcinoma-derived epithelial cells, we investigated how c-lamellipodia form and found that they sporadically grew from around E-cadherin–based adherens junctions (AJs). WAVE and Arp2/3 complexes were localized along the AJs, and silencing them not only interfered with c-lamellipodia formation but also prevented follower cells from trailing the leaders. Disruption of AJs by removing αE-catenin resulted in uncontrolled c-lamellipodia growth, and this was brought about by myosin II activation and the resultant contraction of AJ-associated actomyosin cables. Additional observations indicated that c-lamellipodia tended to grow at mechanically weak sites of the junction. We conclude that AJs not only tie cells together but also support c-lamellipodia formation by recruiting actin regulators, enabling epithelial cells to undergo ordered collective migration.