Insulin stimulates PKCzeta -mediated phosphorylation of insulin receptor substrate-1 (IRS-1). A self-attenuated mechanism to negatively regulate the function of IRS proteins

Incubation of rat hepatoma Fao cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins. This is followed by elevation in their P-Ser/Thr content, and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, abolished the increase in the P-Ser/Thr content of IRS-1, its dissociation from the IR, and the decrease in its P-Tyr content following 60 min of insulin treatment, indicating that the Ser kinases that negatively regulate IRS-1 function are downstream effectors of PI3K. PKCzeta fulfills this criterion, being an insulin-activated downstream effector of PI3K. Overexpression of PKCzeta in Fao cells, by infection of the cells with adenovirus-based PKCzeta construct, had no effect on its own, but it accelerated the rate of insulin-stimulated dissociation of IR.IRS-1 complexes and the rate of Tyr dephosphorylation of IRS-1. The insulin-stimulated negative regulatory role of PKCzeta was specific and could not be mimic by infecting Fao cells with adenoviral constructs encoding for PKC alpha, delta, or eta. Because the reduction in P-Tyr content of IRS-1 was accompanied by a reduced association of IRS-1 with p85, the regulatory subunit of PI3K, it suggests that this negative regulatory process induced by PKCzeta, has a built-in attenuation signal. Hence, insulin triggers a sequential cascade in which PI3K-mediated activation of PKCzeta inhibits IRS-1 functions, reduces complex formation between IRS-1 and PI3K, and inhibits further activation of PKCzeta itself. These findings implicate PKCzeta as a key element in a multistep negative feedback control mechanism of IRS-1 functions.     

Actin cytoskeleton regulates calcium dynamics and NFAT nuclear duration

T-cell activation by antigen-presenting cells is accompanied by actin polymerization, T-cell receptor (TCR) capping, and formation of the immunological synapse. However, whether actin-dependent events are required for T-cell function is poorly understood. Herein, we provide evidence for an unexpected negative regulatory role of the actin cytoskeleton on TCR-induced cytokine production. Disruption of actin polymerization resulted in prolonged intracellular calcium elevation in response to anti-CD3, thapsigargin, or phorbol myristate acetate plus ionomycin, leading to persistent NFAT (nuclear factor of activated T cells) nuclear duration. These events were dominant, as the net effect of actin blockade was augmented interleukin 2 promoter activity. Increased surface expression of the plasma membrane Ca(2+) ATPase was observed upon stimulation, which was inhibited by cytochalasin D, suggesting that actin polymerization contributes to calcium export. Our results imply a novel role for the actin cytoskeleton in modulating the duration of Ca(2+)-NFAT signaling and indicate that actin dynamics regulate features of T-cell activation downstream of receptor clustering.     

Neuregulin signaling on glucose transport in muscle cells

Neuregulin-1, a growth factor that potentiates myogenesis induces glucose transport through translocation of glucose transporters, in an additive manner to insulin, in muscle cells. In this study, we examined the signaling pathway required for a recombinant active neuregulin-1 isoform (rhHeregulin-beta(1), 177-244, HRG) to stimulate glucose uptake in L6E9 myotubes. The stimulatory effect of HRG required binding to ErbB3 in L6E9 myotubes. PI3K activity is required for HRG action in both muscle cells and tissue. In L6E9 myotubes, HRG stimulated PKBalpha, PKBgamma, and PKCzeta activities. TPCK, an inhibitor of PDK1, abolished both HRG- and insulin-induced glucose transport. To assess whether PKB was necessary for the effects of HRG on glucose uptake, cells were infected with adenoviruses encoding dominant negative mutants of PKBalpha. Dominant negative PKB reduced PKB activity and insulin-stimulated glucose transport but not HRG-induced glucose transport. In contrast, transduction of L6E9 myotubes with adenoviruses encoding a dominant negative kinase-inactive PKCzeta abolished both HRG- and insulin-stimulated glucose uptake. In soleus muscle, HRG induced PKCzeta, but not PKB phosphorylation. HRG also stimulated the activity of p70S6K, p38MAPK, and p42/p44MAPK and inhibition of p42/p44MAPK partially repressed HRG action on glucose uptake. HRG did not affect AMPKalpha(1) or AMPKalpha(2) activities. In all, HRG stimulated glucose transport in muscle cells by activation of a pathway that requires PI3K, PDK1, and PKCzeta, but not PKB, and that shows cross-talk with the MAPK pathway. The PI3K, PDK1, and PKCzeta pathway can be considered as an alternative mechanism, independent of insulin, to induce glucose uptake.     

p70 S6 kinase is regulated by protein kinase Czeta and participates in a phosphoinositide 3-kinase-regulated signalling complex

p70 S6 kinase (p70S6K) is an important regulator of cell proliferation. Its activation by growth factor requires phosphorylation by various inputs on multiple sites. Data accumulated thus far support a model whereby p70S6K activation requires sequential phosphorylations at proline-directed residues in the putative autoinhibitory pseudosubstrate domain, as well as threonine 389. Threonine 229, a site in the catalytic loop is phosphorylated by phosphoinositide-dependent kinase 1 (PDK-1). Experimental evidence suggests that p70S6K activation requires a phosphoinositide 3-kinase (PI3-K)-dependent signal(s). However, the intermediates between PI3-K and p70S6K remain unclear. Here, we have identified PI3-K-regulated atypical protein kinase C (PKC) isoform PKCzeta as an upstream regulator of p70S6K. In coexpression experiments, we found that a kinase-inactive PKCzeta mutant antagonized activation of p70S6K by epidermal growth factor, PDK-1, and activated Cdc42 and PI3-K. While overexpression of a constitutively active PKCzeta mutant (myristoylated PKCzeta [myr-PKCzeta]) only modestly activated p70S6K, this mutant cooperated with PDK-1 activation of p70S6K. PDK-1-induced activation of a C-terminal truncation mutant of p70S6K was also enhanced by myr-PKCzeta. Moreover, we have found that p70S6K can associate with both PDK-1 and PKCzeta in vivo in a growth factor-independent manner, while PDK-1 and PKCzeta can also associate with each other, suggesting the existence of a multimeric PI3-K signalling complex. This work provides evidence for a link between a phorbol ester-insensitive PKC isoform and p70S6K. The existence of a PI3-K-dependent signalling complex may enable efficient activation of p70S6K in cells.     

Control sites of ribosomal S6 kinase B and persistent activation through tumor necrosis factor

RSKB, a 90-kDa ribosomal S6 protein kinase family (RSK) member with two complete catalytic domains connected by a linker, is activated through p38- and ERK-mitogen-activated protein kinases. The N-terminal kinases of RSKs phosphorylate substrates; activation requires phosphorylation of linker and C-terminal kinase sites. Unlike other RSKs, the activation loop phosphorylation sites of both catalytic domains of RSKB, Ser(196) and Thr(568), were required for activity. RSKB activation depended on phosphorylation of linker Ser(343) and Ser(360) and associated with phosphorylation of nonconserved Ser(347), but Ser(347)-deficient RSKB retained partial activity. The known protein kinase A and protein kinase C inhibitors, H89 and Ro31-8220, blocked RSKB activity. Treatment of HeLa cells with tumor necrosis factor, epidermal growth factor, phorbol 12-myristate 13-acetate, and ionomycin but not with insulin resulted in strong activation of endogenous RSKB. High RSKB activity and Ser(347)/Ser(360) phosphorylation persisted for 3 h in tumor necrosis factor-treated cells, in contrast to the short bursts of p38, ERK, and RSK1-3 activities. In conclusion, a variety of stimuli induced phosphorylation and activation of RSKB through both p38 and ERK pathways; the persistence of activation indicated that RSKB selectively escaped cell mechanisms causing rapid deactivation of upstream p38 and ERK and other RSKs.     

Phosphorylation of a distinct structural form of phosphatidylinositol transfer protein alpha at Ser166 by protein kinase C disrupts receptor-mediated phospholipase C signaling by inhibiting delivery of phosphatidylinositol to membranes

Phosphatidylinositol transfer protein alpha (PITPalpha) participates in the supply of phosphatidylinositol (PI) required for many cellular events including phospholipase C (PLC) beta and gamma signaling by G-protein-coupled receptors and receptor-tyrosine kinases, respectively. Protein kinase C has been known to modulate PLC signaling by G-protein-coupled receptors and receptor-tyrosine kinases, although the molecular target has not been identified in most instances. In each case phorbol myristate acetate pretreatment of HL60, HeLa, and COS-7 cells abrogated PLC stimulation by the agonists formyl-Met-Leu-Phe, ATP, and epidermal growth factor, respectively. Here we show that phosphorylation of PITPalpha at Ser166 resulted in inhibition of receptor-stimulated PLC activity. Ser166 is localized in a small pocket between the 165-172 loop and the rest of the protein and was not solvent-accessible in either the PI- or phosphatidylcholine-loaded structures of PITPalpha. To allow phosphorylation at Ser166, a distinct structural form is postulated, and mutation of Thr59 to alanine shifted the equilibrium to this form, which could be resolved on native PAGE. The elution profile observed by size exclusion chromatography of phosphorylated PITPalpha from rat brain or in vitro phosphorylated PITPalpha demonstrated that phosphorylated PITPalpha is structurally distinct from the non-phosphorylated form. Phosphorylated PITPalpha was unable to deliver its PI cargo, although it could deliver phosphatidylcholine. We conclude that the PITPalpha structure has to relax to allow access to the Ser166 site, and this may occur at the membrane surface where PI delivery is required for receptor-mediated PLC signaling.     

The bile acid taurochenodeoxycholate activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade

Liver injury during cholestasis reflects a balance between the effects of toxic and nontoxic bile acids. However, the critical distinction between a toxic and nontoxic bile acid remains subtle and unclear. For example, the glycine conjugate of chenodeoxycholate (GCDC) induces hepatocyte apoptosis, whereas the taurine conjugate (TCDC) does not. We hypothesized that the dissimilar cellular responses may reflect differential activation of a phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway. In the bile acid-transporting McNtcp.24 rat hepatoma cell line, TCDC, but not GCDC, stimulated PI3K activity. Consistent with this observation, inhibition of PI3K rendered TCDC cytotoxic, and constitutive activation of PI3K rendered GCDC nontoxic. Both Akt and the atypical protein kinase C isoform zeta (PKCzeta) have been implicated in PI3K-dependent survival signaling. However, TCDC activated PKCzeta, but not Akt. Moreover, inhibition of PKCzeta converted TCDC into a cytotoxic agent, whereas overexpression of wild-type PKCzeta blocked GCDC-induced apoptosis. We also demonstrate that TCDC activated nuclear factor kappaB (NF-kappaB) in a PI3K- and PKCzeta-dependent manner. Moreover, inhibition of NF-kappaB by an IkappaB super-repressor rendered TCDC cytotoxic, suggesting that NF-kappaB is also necessary to prevent the cytotoxic effects of TCDC. Collectively, these data suggest that some hydrophobic bile acids such as TCDC activate PI3K-dependent survival pathways, which prevent their otherwise inherent toxicity.     

Activation of mitogen-activated protein kinases is required for alpha1-adrenergic agonist-induced cell scattering in transfected HepG2 cells

Activation of alpha1B-adrenergic receptors ((alpha1B)AR) by phenylephrine (PE) induces scattering of HepG2 cells stably transfected with the (alpha1B)AR (TFG2 cells). Scattering was also observed after stimulation of TFG2 cells with phorbol myristate acetate (PMA) but not with hepatocyte growth factor/scatter factor, epidermal growth factor, or insulin. PMA but not phenylephrine rapidly activated PKCalpha in TFG2 cells, and the highly selective PKC inhibitor bisindolylmaleimide (GFX) completely abolished PMA-induced but not PE-induced scattering. PE rapidly activated p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK, c-Jun N-terminal kinase (JNK), and AP1 (c-fos/c-jun). Selective blockade of p42/44 MAPK activity by PD98059 or by transfection of a MEK1 dominant negative adenovirus significantly inhibited the PE-induced scattering of TFG2 cells. Selective inhibition of p38 MAPK by SB203850 or SB202190 also blocked PE-induced scattering, whereas treatment of TFG2 cells with the PI3 kinase inhibitors LY294002 or wortmannin did not inhibit PE-induced scattering. Blocking JNK activation with a dominant negative mutant of JNK or blocking AP1 activation with a dominant negative mutant of c-jun (TAM67) significantly inhibited PE-induced cell scattering. These data indicate that PE-induced scattering of TFG2 cells is mediated by complex mechanisms, including activation of p42/44 MAPK, p38 MAPK, and JNK. Cell spreading has been reported to play important roles in wound repair, tumor invasion, and metastasis. Therefore, catecholamines acting via the (alpha1)AR may modulate these physiological and pathological processes.     

Immune complex-induced integrin activation and L-plastin phosphorylation require protein kinase A.

Integrins in resting leukocytes are poorly adhesive, and cell activation is required to induce integrin-mediated adhesion. We recently demonstrated a close correlation between phosphorylation of Ser(5) in L-plastin (LPL), a leukocyte-specific 67-kDa actin bundling protein, and activation of alpha(M)beta(2)-mediated adhesion in polymorphonuclear neutrophils (PMN) (Jones, S. L., Wang, J., Turck, C. W., and Brown, E. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9331-9336). However, the kinase that phosphorylates LPL Ser(5) has not been identified. We found that cAMP-dependent protein kinase (PKA), but not a variety of other serine kinases, can specifically phosphorylate LPL and LPL-derived peptides on Ser(5) in vitro. The cell-permeable cAMP analog 8-bromo-cAMP and the adenylate cyclase activator forskolin both induce LPL phosphorylation in cells. Two PKA inhibitors, H89 and KT5720, inhibited immune complex (IC)-stimulated LPL phosphorylation as well as IC-induced activation of alpha(M)beta(2)-mediated adhesion in PMN. The dose response of H89 inhibition of PMN adhesion correlated with its inhibition of LPL phosphorylation in response to IC. IC stimulation also transiently increased intracellular cAMP concentration in PMN. Thus, PKA functions in an integrin activation pathway initiated by IC binding to Fcgamma receptors in addition to its better known role as a negative regulator of cell activation by G protein-coupled receptors. In contrast, LPL Ser(5) phosphorylation and PMN adhesion induced by formylmethionyl-leucylphenylalanine or phorbol myristate acetate were not affected by PKA inhibitors, suggesting that a different kinase(s) is responsible for LPL phosphorylation in response to these agonists. Phosphoinositidyl 3-kinase also is required for FcgammaR but not formylmethionyl-leucylphenylalanine- or phorbol myristate acetate-induced LPL phosphorylation and activation of alpha(M)beta(2). Two phosphoinositidyl 3-kinase inhibitors blocked FcgammaR-induced cAMP accumulation, demonstrating that this kinase acts upstream of PKA. These data demonstrate a necessary role for PKA in IC-induced integrin activation and LPL phosphorylation.     

Immunosuppressive properties of synthetic peptides derived from CD4 and HLA-DR antigens

Synthetic peptides derived from the beta 1 domain of HLA-DR antigens containing RFDS and a peptide derived from the immunoglobulin-like amino-terminal domain of CD4 and containing RADS were shown to exhibit specific dose-dependent inhibitory effects on antigen-induced HLA class II-restricted T-cell proliferation and in vitro antibody synthesis. These inhibitory activities are similar to those exhibited by anti-CD4 and HLA-DR antibodies, respectively. The peptides derived from HLA-DR or CD4 and anti-CD4 or anti-HLA-DR antibodies acted together in synergy to inhibit these responses when the relevant cell populations were incubated with infrainhibitory concentrations of the reagents. In contrast, these peptides were shown to exert no inhibitory activity on nonspecific T-cell activation mediated by ionomycin, phorbol myristate acetate, and interleukin-2.