Hsp90 regulates p50(cdc37) function during the biogenesis of the activeconformation of the heme-regulated eIF2 alpha kinase

Recent studies indicate that p50(cdc37) facilitates Hsp90-mediated biogenesis of certain protein kinases. In this report, we examined whether p50(cdc37) is required for the biogenesis of the heme-regulated eIF2 alpha kinase (HRI) in reticulocyte lysate. p50(cdc37) interacted with nascent HRI co-translationally and this interaction persisted during the maturation and activation of HRI. p50(cdc37) stimulated HRI's activation in response to heme deficiency, but did not activate HRI per se. p50(cdc37) function was specific to immature and inactive forms of the kinase. Analysis of mutant Cdc37 gene products indicated that the N-terminal portion of p50(cdc37) interacted with immature HRI, but not with Hsp90, while the C-terminal portion of p50(cdc37) interacted with Hsp90. The Hsp90-specific inhibitor geldanamycin disrupted the ability of both Hsp90 and p50(cdc37) to bind HRI and promote its activation, but did not disrupt the native association of p50(cdc37) with Hsp90. A C-terminal truncated mutant of p50(cdc37) inhibited HRI's activation, prevented the interaction of Hsp90 with HRI, and bound to HRI irrespective of geldanamycin treatment. Additionally, native complexes of HRI with p50(cdc37) were detected in cultured K562 erythroleukemia cells. These results suggest that p50(cdc37) provides an activity essential to HRI biogenesis via a process regulated by nucleotide-mediated conformational switching of its partner Hsp90.     

Involvement of heat shock protein (Hsp)90 beta but not Hsp90 alpha in antiapoptotic effect of CpG-B oligodeoxynucleotide

Unmethylated CpG oligodeoxynucleotides (CpG ODNs) activate immune responses in a TLR9-dependent manner. In this study, we found that stimulation of mouse macrophages and dendritic cells with B-type CpG ODN (CpG-B ODN) increased the cellular level of heat shock protein (Hsp) 90beta but not Hsp90alpha and prevented apoptosis induced by serum starvation or staurosporine treatment. The CpG-B ODN-induced Hsp90beta expression depended on TLR9, MyD88, and PI3K. Inhibition of Hsp90beta level by expressing small-interfering RNA suppressed not only Hsp90beta expression but also PI3K-dependent phosphorylation of Akt and CpG-B ODN-mediated antiapoptosis. Additional studies demonstrated that as described by other group in mast cells, Hsp90beta but not Hsp90alpha was associated with Bcl-2. Inhibition of Hsp90beta suppressed the CpG-B ODN-induced association of Hsp90beta with Bcl-2 and impaired the inhibitory effect of CpG-B ODN in the release of cytochrome c and activation of caspase-3. This study thus reveals the involvement of Hsp90beta but not Hsp90alpha in CpG-B ODN-mediated antiapoptotic response and that Hsp90beta is distinct from Hsp90alpha in regulation of the cellular function of immune cells.     

Dual lipid modification motifs in G(alpha) and G(gamma) subunits are required for full activity of the pheromone response pathway in Saccharomyces cerevisiae

To establish the biological function of thioacylation (palmitoylation), we have studied the heterotrimeric guanine nucleotide-binding protein (G protein) subunits of the pheromone response pathway of Saccharomyces cerevisiae. The yeast G protein gamma subunit (Ste18p) is unusual among G(gamma) subunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106. Substitution of either cysteine results in a strong signaling defect. In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107. Ste18p was targeted to the plasma membrane even in the absence of prenylation or thioacylation. However, G protein activation released prenylation- or thioacylation-defective Ste18p into the cytoplasm. Hence, lipid modifications of the G(gamma) subunit are dispensable for G protein activation by receptor, but they are required to maintain the plasma membrane association of G(betagamma) after receptor-stimulated release from G(alpha). The G protein alpha subunit (Gpa1p) is tandemly modified at its N terminus with amide- and thioester-linked fatty acids. Here we show that Gpa1p was thioacylated in vivo with a mixture of radioactive myristate and palmitate. Mutation of the thioacylation site in Gpa1p resulted in yeast cells that displayed partial activation of the pathway in the absence of pheromone. Thus, dual lipidation motifs on Gpa1p and Ste18p are required for a fully functional pheromone response pathway.     

Calcium channel beta subunit promotes voltage-dependent modulation of alpha 1 B by G beta gamma

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming alpha1 subunit and auxiliary subunits, including the intracellular beta subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G beta gamma dimer concentration. Here we have examined the interaction between the regulation of N-type (alpha 1 B) channels by their beta subunits and by G beta gamma dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC beta subunit, we found a significantly larger G beta gamma-dependent inhibition of alpha 1 B channel activation when the VDCC alpha 1 B and beta subunits were coexpressed. In the absence of coexpressed VDCC beta subunit, the G beta gamma dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC beta subunit coexpression restored all of the hallmarks of G beta gamma modulation. These results suggest that the VDCC beta subunit must be present for G beta gamma to induce voltage-dependent modulation of N-type calcium channels.     

Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin alpha IIbbeta 3 activation in platelets

Platelet activation is a complex process induced by a variety of stimuli, which act in concert to ensure the rapid formation of a platelet plug at places of vascular injury. We show here that fibrillar collagen, which initiates platelet activation at the damaged vessel wall, activates only a small fraction of platelets in suspension directly, whereas the majority of platelets becomes activated by mediators released from collagen-activated platelets. In Galpha(q)-deficient platelets that do not respond with activation of integrin alpha(IIb)beta(3) to a variety of mediators like thromboxane A2 (TXA2), thrombin, or ADP, collagen at high concentrations was able to induce aggregation, an effect that could be blocked by antagonists of the TXA2 or P2Y12 receptors. The activation of TXA2 or P2Y12 receptors alone, which in Galpha(q)-deficient platelets couple to G12/G13 and Gi, respectively, did not induce platelet integrin activation or aggregation. However, concomitant activation of both receptors resulted in irreversible integrin alpha(IIb)beta3-mediated aggregation of Galpha(q)-deficient platelets. Thus, the activation of G12/G13- and Gi-mediated signaling pathways is sufficient to induce integrin alpha(IIb)beta3 activation. Although G(q)-mediated signaling plays an important role in platelet activation, it is not strictly required for the activation of integrin alpha(IIb)beta3. This indicates that the efficient induction of platelet aggregation through G-protein-coupled receptors is an integrated response mediated by various converging G-protein-mediated signaling pathways involving G(q) and G(i) as well as G12/G13.     

Chimeric G alpha i2/G alpha 13 proteins reveal the structural requirements for the binding and activation of the RGS-like (RGL)-containing Rho guanine nucleotide exchange factors (GEFs) by G alpha 13

The alpha-subunit of G proteins of the G(12/13) family stimulate Rho by their direct binding to the RGS-like (RGL) domain of a family of Rho guanine nucleotide exchange factors (RGL-RhoGEFs) that includes PDZ-RhoGEF (PRG), p115RhoGEF, and LARG, thereby regulating cellular functions as diverse as shape and movement, gene expression, and normal and aberrant cell growth. The structural features determining the ability of G alpha(12/13) to bind RGL domains and the mechanism by which this association results in the activation of RGL-RhoGEFs are still poorly understood. Here, we explored the structural requirements for the functional interaction between G alpha(13) and RGL-RhoGEFs based on the structure of RGL domains and their similarity with the area by which RGS4 binds the switch region of G alpha(i) proteins. Using G alpha(i2), which does not bind RGL domains, as the backbone in which G alpha(13) sequences were swapped or mutated, we observed that the switch region of G alpha(13) is strictly necessary to bind PRG, and specific residues were identified that are critical for this association, likely by contributing to the binding surface. Surprisingly, the switch region of G alpha(13) was not sufficient to bind RGL domains, but instead most of its GTPase domain is required. Furthermore, membrane localization of G alpha(13) and chimeric G alpha(i2) proteins was also necessary for Rho activation. These findings revealed the structural features by which G alpha(13) interacts with RGL domains and suggest that molecular interactions occurring at the level of the plasma membrane are required for the functional activation of the RGL-containing family of RhoGEFs.     

G13alpha-mediated PYK2 activation. PYK2 is a mediator of G13alpha -induced serum response element-dependent transcription

G(12)alpha/G(13)alpha transduces signals from G-protein-coupled receptors to stimulate growth-promoting pathways and the early response gene c-fos. Within the c-fos promoter lies a key regulatory site, the serum response element (SRE). Here we show a critical role for the tyrosine kinase PYK2 in muscarinic receptor type 1 and G(12)alpha/G(13)alpha signaling to an SRE reporter gene. A kinase-inactivate form of PYK2 (PYK2 KD) inhibits muscarinic receptor type 1 signaling to the SRE and PYK2 itself triggers SRE reporter gene activation through a RhoA-dependent pathway. Placing PYK2 downstream of G-protein activation but upstream of RhoA, the expression of PYK2 KD blocks the activation of an SRE reporter gene by GTPase-deficient forms of G(12)alpha or G(13)alpha but not by RhoA. The GTPase-deficient form of G(13)alpha triggers PYK2 kinase activity and PYK2 tyrosine phosphorylation, and co-expression of the RGS domain of p115 RhoGEF inhibits both responses. Finally, we show that in vivo G(13)alpha, although not G(12)alpha, readily associates with PYK2. Thus, G-protein-coupled receptors via G(13)alpha activation can use PYK2 to link to SRE-dependent gene expression.     

G Protein activation without subunit dissociation depends on a G{alpha}(i)-specific region

G proteins transmit a variety of extracellular signals into intracellular responses. The Galpha and Gbetagamma subunits are both known to regulate effectors. Interestingly, the Galpha subunit also determines subtype specificity of Gbetagamma effector interactions. However, in light of the common paradigm that Galpha and Gbetagamma subunits dissociate during activation, a plausible mechanism of how this subtype specificity is generated was lacking. Using a fluorescence resonance energy transfer (FRET)-based assay developed to directly measure mammalian G protein activation in intact cells, we demonstrate that fluorescent Galpha(i1,2,3), Galpha(z), and Gbeta(1)gamma(2) subunits do not dissociate during activation but rather undergo subunit rearrangement as indicated by an activation-induced increase in FRET. In contrast, fluorescent Galpha(o) subunits exhibited an activation-induced decrease in FRET, reflecting subunit dissociation or, alternatively, a distinct subunit rearrangement. The alpha(B/C)-region within the alpha-helical domain, which is much more conserved within Galpha(i1,2,3) and Galpha(z) as compared with that in Galpha(o), was found to be required for exhibition of an activation-induced increase in FRET between fluorescent Galpha and Gbetagamma subunits. However, the alpha(B/C)-region of Galpha(il) alone was not sufficient to transfer the activation pattern of Galpha(i) to the Galpha(o) subunit. Either residues in the first 91 amino acids or in the C-terminal remainder (amino acids 93-354) of Galpha(il) together with the alpha(B/C)-helical region of Galpha(i1) were needed to transform the Galpha(o)-activation pattern into a Galpha(i1)-type of activation. The discovery of subtype-selective mechanisms of G protein activation illustrates that G protein subfamilies have specific mechanisms of activation that may provide a previously unknown basis for G protein signaling specificity.     

Constitutively active alpha subunits of G(q/11) and G(12/13) families inhibit activation of the pro-survival Akt signaling cascade

Accumulating evidence indicates that G protein signaling plays an active role in the regulation of cell survival. Our previous study demonstrated the regulatory effects of G(i/o) proteins in nerve growth factor-induced activation of pro-survival Akt kinase. In the present study we explored the role of various members of the G(s), G(q/11) and G(12/13) subfamilies in the regulation of Akt in cultured mammalian cells. In human embryonic kidney 293 cells transiently expressing constitutively active mutants of G alpha11, G alpha14, G alpha16, G alpha12, or G alpha13 (G alpha11QL, G alpha14QL, G alpha16QL, G alpha12QL and G alpha13QL, respectively), basal phosphorylation of Akt was attenuated, as revealed by western blotting analysis using a phosphospecific anti-Akt immunoglobulin. In contrast, basal Akt phosphorylation was unaffected by the overexpression of a constitutively active G alpha(s) mutant (G alpha(s)QL). Additional experiments showed that G alpha11QL, G alpha14QL, G alpha16QL, G alpha12QL and G alpha13QL, but not G alpha(s)QL, attenuated phosphorylation of the Akt-regulated translation regulator tuberin. Moreover, they were able to inhibit the epidermal growth factor-induced Akt activation and tuberin phosphorylation. The inhibitory mechanism of Gq family members was independent of phospholipase Cbeta activation and calcium signaling because G alpha11QL, G alpha14QL and G alpha16QL remained capable of inhibiting epidermal growth factor-induced Akt activation in cells pretreated with U73122 and the intracellular calcium chelator, BAPTA/AM. Finally, overexpression of the dominant negative mutant of RhoA blocked G alpha12QL- and G alpha13QL-mediated inhibition, suggesting that activated G alpha12 and G alpha13 inhibit Akt signaling via RhoA. Collectively, this study demonstrated the inhibitory effect of activated G alpha11, G alpha14, G alpha16, G alpha12 and G alpha13 on pro-survival Akt signaling.     

The trimeric GTP-binding protein (G(q)/G(11)) alpha subunit is required for insulin-stimulated GLUT4 translocation in 3T3L1 adipocytes

To investigate the potential role of trimeric GTP-binding proteins regulating GLUT4 translocation in adipocytes, wild type and constitutively active G(q) (G(q)/Q209L), G(i) (G(i)/Q205L), and G(s) (G(s)/Q227L) alpha subunit mutants were expressed in 3T3L1 adipocytes. Although expression of neither the wild type nor G(i)/Q205L and G(s)/Q227L alpha subunit mutants had any effect on the basal or insulin-stimulated translocation of a co-expressed GLUT4-enhanced green fluorescent protein (EGFP) fusion protein, expression of G(q)/Q209L resulted in GLUT4-EGFP translocation in the absence of insulin. In contrast, microinjection of an inhibitory G(q)/G(11) alpha subunit-specific antibody but not a G(i) or G(s) alpha subunit antibody prevented insulin-stimulated endogenous GLUT4 translocation. Consistent with a required role for GTP-bound G(q)/G(11), expression of the regulators of G protein signaling (RGS4 and RGS16) also attenuated insulin-stimulated GLUT4-EGFP translocation. To assess the relationship between G(q)/G(11) function with the phosphatidylinositol 3-kinase dependent pathway, expression of a dominant-interfering p85 regulatory subunit, as well as wortmannin treatment inhibited insulin-stimulated but not G(q)/Q209L-stimulated GLUT4-EGFP translocation. Furthermore, G(q)/Q209L did not induce the in vivo accumulation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), whereas expression of the RGS proteins did not prevent the insulin-stimulated accumulation of PIP(3). Together, these data demonstrate that insulin stimulation of GLUT4 translocation requires at least two independent signal transduction pathways, one mediated through the phosphatidylinositol 3-kinase and another through the trimeric GTP-binding proteins G(q) and/or G(11).     

JlpA of Campylobacter jejuni interacts with surface-exposed heat shock protein 90alpha and triggers signalling pathways leading to the activation of NF-kappaB and p38 MAP kinase in epithelial cells

Campylobacter jejuni is a leading cause of acute bacterial gastroenteritis in humans. The mechanism by which C. jejuni interacts with host cells, however, is still poorly understood. Our previous study has shown that the C. jejuni surface lipoprotein JlpA mediates adherence of the bacterium to epithelial cells. In this report, we demonstrated that JlpA interacts with HEp-2 cell surface heat shock protein (Hsp) 90alpha and initiates signalling pathways leading to activation of NF-kappaB and p38 MAP kinase. Gel overlay and GST pull down assays showed that JlpA interacts with Hsp90alpha. Geldanamycin, a specific inhibitor of Hsp90, and anti-human Hsp90alpha antibody significantly blocked the interaction between JlpA and Hsp90alpha, suggesting a direct interaction between JlpA and HEp-2 cell surface-exposed Hsp90alpha. The treatment of HEp-2 cells with GST-JlpA initiated two signalling pathways: one leading to the phosphorylation and degradation of IkappaB and nuclear translocation of NF-kappaB; and another one to the phosphorylation of p38 MAP kinase. The activation of NF-kappaB and p38 MAP kinase in HEp-2 cells suggest that JlpA triggers inflammatory/immune responses in host cells following C. jejuni infection.     

Regulation of cardiac L-type Ca2+ channel by coexpression of G(alpha s) in Xenopus oocytes

Activation of G(alpha s) via beta-adrenergic receptors enhances the activity of cardiac voltage-dependent Ca2+ channels of the L-type, mainly via protein kinase A (PKA)-dependent phosphorylation. Contribution of a PKA-independent effect of G(alpha s) has been proposed but remains controversial. We demonstrate that, in Xenopus oocytes, antisense knockdown of endogenous G(alpha s) reduced, whereas coexpression of G(alpha s) enhanced, currents via expressed cardiac L-type channels, independently of the presence of the auxiliary subunits alpha2/delta or beta2A. Coexpression of G(alpha s) did not increase the amount of alpha1C protein in whole oocytes or in the plasma membrane (measured immunochemically). Activation of coexpressed beta2 adrenergic receptors did not cause a detectable enhancement of channel activity; rather, a small cAMP-dependent decrease was observed. We conclude that coexpression of G(alpha s), but not its acute activation via beta-adrenergic receptors, enhances the activity of the cardiac L-type Ca2+ channel via a PKA-independent effect on the alpha1C subunit.     

Interaction of heterotrimeric G13 protein with an A-kinase-anchoring protein 110 (AKAP110) mediates cAMP-independent PKA activation.

Heterotrimeric G proteins and protein kinase A (PKA) are two important transmitters that transfer signals from a wide variety of cell surface receptors to generate physiological responses. The established mechanism of PKA activation involves the activation of the Gs-cAMP pathway. Binding of cAMP to the regulatory subunit of PKA (rPKA) leads to a release and subsequent activation of a catalytic subunit of PKA (cPKA). Here, we report a novel mechanism of PKA stimulation that does not require cAMP. Using yeast two-hybrid screening, we found that the alpha subunit of G13 protein interacted with a member of the PKA-anchoring protein family, AKAP110. Using in vitro binding and coimmunoprecipitation assays, we have shown that only activated G alpha 13 binds to AKAP110, suggesting a potential role for AKAP110 as a G alpha subunit effector protein. Importantly, G alpha 13, AKAP110, rPKA, and cPKA can form a complex, as shown by coimmunoprecipitation. By characterizing the functional significance of the G alpha 13-AKAP110 interaction, we have found that G alpha 13 induced release of the cPKA from the AKAP110-rPKA complex, resulting in a cAMP-independent PKA activation. Finally, AKAP110 significantly potentiated G alpha 13-induced activation of PKA. Thus, AKAP110 provides a link between heterotrimeric G proteins and cAMP-independent activation of PKA.     

GTPase-deficient G alpha 16 and G alpha q induce PC12 cell differentiation and persistent activation of cJun NH2-terminal kinases.

Persistent stimulation of specific protein kinase pathways has been proposed as a key feature of receptor tyrosine kinases and intracellular oncoproteins that signal neuronal differentiation of rat pheochromocytoma (PC12) cells. Among the protein serine/threonine kinases identified to date, the p42/44 mitogen-activated protein (MAP) kinases have been highlighted for their potential role in signalling PC12 cell differentiation. We report here that retrovirus-mediated expression of GTPase-deficient, constitutively active forms of the heterotrimeric Gq family members, G alpha qQ209L and G alpha 16Q212L, in PC12 cells induces neuronal differentiation as indicated by neurite outgrowth and the increased expression of voltage-dependent sodium channels. Differentiation was not observed after cellular expression of GTPase-deficient forms of alpha i2 or alpha 0, indicating selectivity for the Gq family of G proteins. As predicted, overexpression of alpha qQ209L and alpha 16Q212L constitutively elevated basal phospholipase C activity approximately 10-fold in PC12 cells. Significantly, little or no p42/44 MAP kinase activity was detected in PC12 cells differentiated with alpha 16Q212L or alpha qQ209L, although these proteins were strongly activated following expression of constitutively active cRaf-1. Rather, a persistent threefold activation of the cJun NH2-terminal kinases (JNKs) was observed in PC12 cells expressing alpha qQ209L and alpha 16Q212L. This level of JNK activation was similar to that achieved with nerve growth factor, a strong inducer of PC12 cell differentiation. Supportive of a role for JNK activation in PC12 cell differentiation, retrovirus-mediated overexpression of cJun, a JNK target, in PC12 cells induced neurite outgrowth. The results define a p42/44 MAP kinase-independent mechanism for differentiation of PC12 cells and suggest that persistent activation of the JNK members of the proline-directed protein kinase family by GTPase-deficient G alpha q and G alpha 16 subunits is sufficient to induce differentiation of PC12 cells.     

Rhodopsin recognition by mutant G(s)alpha containing C-terminal residues of transducin

The C-terminal regions of the heterotrimeric G protein alpha-subunits play key roles in selective activation of G proteins by their cognate receptors. In this study, mutant G(s)alpha proteins with substitutions by C-terminal residues of transducin (G(t)alpha) were analyzed for their interaction with light-activated rhodopsin (R*) to delineate the critical determinants of the G(t)alpha/R* coupling. In contrast to G(s)alpha, a chimeric G(s)alpha/G(t)alpha protein containing only 11 C-terminal residues from transducin was capable of binding to and being potently activated by R*. Our results suggest that Cys(347) and Gly(348) are absolutely essential, whereas Asp(346) is more modestly involved in the G(t) activation by R*. In addition, the analysis of the intrinsic nucleotide exchange in mutant G(s)alpha indicated an interaction between the C terminus and the switch II region in G(t)alpha.GDP. Mutant G(s)alpha containing the G(t)alpha C terminus and substitutions of Asn(239) and Asp(240) (switch II) by the corresponding G(t)alpha residues, Glu(212) and Gly(213), displayed significant reductions in spontaneous guanosine 5'-O-(3-thiotriphosphate)-binding rates to the levels approaching those in G(t)alpha. Communication between the C terminus and switch II of G(t)alpha does not appear essential for the activational coupling between G(t) and R*, but may represent one of the mechanisms by which Galpha subunits control intrinsic nucleotide exchange.     

Inhibition of G alpha i2 activation by G alpha i3 in CXCR3-mediated signaling

G protein-coupled receptors (GPCRs) convey extracellular stimulation into dynamic intracellular action, leading to the regulation of cell migration and differentiation. T lymphocytes express G alpha(i2) and G alpha(i3), two members of the G alpha(i/o) protein family, but whether these two G alpha(i) proteins have distinguishable roles guiding T cell migration remains largely unknown because of a lack of member-specific inhibitors. This study details distinct G alpha(i2) and G alpha(i3) effects on chemokine receptor CXCR3-mediated signaling. Our data showed that G alpha(i2) was indispensable for T cell responses to three CXCR3 ligands, CXCL9, CXCL10, and CXCL11, as the lack of G alpha(i2) abolished CXCR3-stimulated migration and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) incorporation. In sharp contrast, T cells isolated from G alpha(i3) knock-out mice displayed a significant increase in both GTPgammaS incorporation and migration as compared with wild type T cells when stimulated with CXCR3 agonists. The increased GTPgammaS incorporation was blocked by G alpha(i3) protein in a dose-dependent manner. G alpha(i3)-mediated blockade of G alpha(i2) activation did not result from G alpha(i3) activation, but instead resulted from competition or steric hindrance of G alpha(i2) interaction with the CXCR3 receptor via the N terminus of the second intracellular loop. A mutation in this domain abrogated not only G alpha(i2) activation induced by a CXCR3 agonist but also the interaction of G alpha(i3) to the CXCR3 receptor. These findings reveal for the first time an interplay of G alpha(i) proteins in transmitting G protein-coupled receptor signals. This interplay has heretofore been masked by the use of pertussis toxin, a broad inhibitor of the G alpha(i/o) protein family.