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1.
We have recently identified WDR26 as a novel WD40 repeat protein that binds Gβγ and promotes Gβγ signaling during leukocyte migration. Here, we have determined the mechanism by which WDR26 enhances Gβγ-mediated phospholipase C β2 (PLCβ2) activation in leukocytes. We show that WDR26 not only directly bound Gβγ but also PLCβ2. The binding sites of WDR26 and PLCβ2 on Gβ1γ2 were overlapping but not identical. WDR26 used the same domains for binding Gβγ and PLCβ but still formed a signaling complex with Gβγ and PLCβ2 probably due to the fact that WDR26 formed a higher order oligomer through its Lis homology and C-terminal to LisH (LisH-CTLH) and WD40 domains. Additional studies indicated that the formation of higher order oligomers was required for WDR26 to promote PLCβ2 interaction with and activation by Gβγ. Moreover, WDR26 was required for PLCβ2 translocation from the cytosol to the membrane in polarized leukocytes, and the translocation of PLCβ2 was sufficient to cause partial activation of PLCβ2. Collectively, our data indicate that WDR26 functions as a scaffolding protein to promote PLCβ2 membrane translocation and interaction with Gβγ, thereby enhancing PLCβ2 activation in leukocytes. These findings have identified a novel mechanism of regulating Gβγ signaling through a scaffolding protein.  相似文献   

2.
Extra-large stimulatory Gα (XLαs) is a large variant of G protein αs subunit (Gαs) that uses an alternative promoter and thus differs from Gαs at the first exon. XLαs activation by G protein–coupled receptors mediates cAMP generation, similarly to Gαs; however, Gαs and XLαs have been shown to have distinct cellular and physiological functions. For example, previous work suggests that XLαs can stimulate inositol phosphate production in renal proximal tubules and thereby regulate serum phosphate levels. In this study, we show that XLαs directly and specifically stimulates a specific isoform of phospholipase Cβ (PLCβ), PLCβ4, both in transfected cells and with purified protein components. We demonstrate that neither the ability of XLαs to activate cAMP generation nor the canonical G protein switch II regions are required for PLCβ stimulation. Furthermore, this activation is nucleotide independent but is inhibited by Gβγ, suggesting a mechanism of activation that relies on Gβγ subunit dissociation. Surprisingly, our results indicate that enhanced membrane targeting of XLαs relative to Gαs confers the ability to activate PLCβ4. We also show that PLCβ4 is required for isoproterenol-induced inositol phosphate accumulation in osteocyte-like Ocy454 cells. Taken together, we demonstrate a novel mechanism for activation of phosphoinositide turnover downstream of Gs-coupled receptors that may have a critical role in endocrine physiology.  相似文献   

3.
L-type voltage dependent Ca2+ channels (L-VDCCs; Cav1.2) are crucial in cardiovascular physiology. In heart and smooth muscle, hormones and transmitters operating via Gq enhance L-VDCC currents via essential protein kinase C (PKC) involvement. Heterologous reconstitution studies in Xenopus oocytes suggested that PKC and Gq-coupled receptors increased L-VDCC currents only in cardiac long N-terminus (NT) isoforms of α1C, whereas known smooth muscle short-NT isoforms were inhibited by PKC and Gq activators. We report a novel regulation of the long-NT α1C isoform by Gβγ. Gβγ inhibited whereas a Gβγ scavenger protein augmented the Gq- but not phorbol ester-mediated enhancement of channel activity, suggesting that Gβγ acts upstream from PKC. In vitro binding experiments reveal binding of both Gβγ and PKC to α1C-NT. However, PKC modulation was not altered by mutations of multiple potential phosphorylation sites in the NT, and was attenuated by a mutation of C-terminally located serine S1928. The insertion of exon 9a in intracellular loop 1 rendered the short-NT α1C sensitive to PKC stimulation and to Gβγ scavenging. Our results suggest a complex antagonistic interplay between Gq-activated PKC and Gβγ in regulation of L-VDCC, in which multiple cytosolic segments of α1C are involved.  相似文献   

4.
Physiologically, only a few primordial follicles are activated to enter the growing follicle pool each wave. Recent studies in knock-out mice show that early follicular activation depends on signaling from the tuberous sclerosis complex, the mammalian target of rapamycin complex 1 (mTORC1), phosphatase and tensin homolog deleted on chromosome 10, and phosphatidylinositol 3-kinase (PI3K) pathways. However, the manner in which these pathways are normally regulated, and whether or not TGF-β acts on them are poorly understood. So, this study aims to identify whether or not TGF-β acts on the process. Ovary organ culture experiments showed that the culture of 18.5 days post-coitus (dpc) ovaries with TGF-β1 reduced the total population of oocytes and activated follicles, accelerated oocyte growth was observed in ovaries treated with TGF-βR1 inhibitor 2-(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-yl-amine (SD208) compared with control ovaries, the down-regulation of TGFR1 gene expression also activated early primordial follicle oocyte growth. We further showed that there was dramatically more proliferation of granulosa cells in SD208-treated ovaries and less proliferation in TGF-β1-treated ovaries. Western blot and morphological analyses indicated that TGF-β signaling manipulated primordial follicle growth through tuberous sclerosis complex/mTORC1 signaling in oocytes, and the mTORC1-specific inhibitor rapamycin could partially reverse the stimulated effect of SD208 on the oocyte growth and decreased the numbers of growing follicles. In conclusion, our results suggest that TGF-β signaling plays an important physiological role in the maintenance of the dormant pool of primordial follicles, which functions through activation of p70 S6 kinase 1 (S6K1)/ribosomal protein S6 (rpS6) signaling in mouse ovaries.  相似文献   

5.
IL-32α is known as a proinflammatory cytokine. However, several evidences implying its action in cells have been recently reported. In this study, we present for the first time that IL-32α plays an intracellular mediatory role in IL-6 production using constitutive expression systems for IL-32α in THP-1 cells. We show that phorbol 12-myristate 13-acetate (PMA)-induced increase in IL-6 production by IL-32α-expressing cells was higher than that by empty vector-expressing cells and that this increase occurred in a time- and dose-dependent manner. Treatment with MAPK inhibitors did not diminish this effect of IL-32α, and NF-κB signaling activity was similar in the two cell lines. Because the augmenting effect of IL-32α was dependent on the PKC activator PMA, we tested various PKC inhibitors. The pan-PKC inhibitor Gö6850 and the PKCϵ inhibitor Ro-31-8220 abrogated the augmenting effect of IL-32α on IL-6 production, whereas the classical PKC inhibitor Gö6976 and the PKCδ inhibitor rottlerin did not. In addition, IL-32α was co-immunoprecipitated with PMA-activated PKCϵ, and this interaction was totally inhibited by the PKCϵ inhibitor Ro-31-8220. PMA-induced enhancement of STAT3 phosphorylation was observed only in IL-32α-expressing cells, and this enhancement was inhibited by Ro-31-8220, but not by Gö6976. We demonstrate that IL-32α mediated STAT3 phosphorylation by forming a trimeric complex with PKCϵ and enhanced STAT3 localization onto the IL-6 promoter and thereby increased IL-6 expression. Thus, our data indicate that the intracellular interaction of IL-32α with PKCϵ and STAT3 promotes STAT3 binding to the IL-6 promoter by enforcing STAT3 phosphorylation, which results in increased production of IL-6.  相似文献   

6.
Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, Gq, G12 and Gi. PMT has been shown by others to lead to the deamidation of recombinant Gαi at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native Gα subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the Gα subunit of the Gq and Gi families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these Gα subunits. Surprisingly, PMT also induced a similar modification of Gα11, a member of the Gq family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of Gα13 was observed following PMT treatment of cells, suggesting differential modification of this Gα subunit by PMT. Gs was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated Gαi, but not Gαq. We also show that PMT inhibits the GTPase activity of Gq.  相似文献   

7.
During signal transduction, the G protein, Gαq, binds and activates phospholipase C-β isozymes. Several diseases have been shown to manifest upon constitutively activating mutation of Gαq, such as uveal melanoma. Therefore, methods are needed to directly inhibit Gαq. Previously, we demonstrated that a peptide derived from a helix-turn-helix (HTH) region of PLC-β3 (residues 852–878) binds Gαq with low micromolar affinity and inhibits Gαq by competing with full-length PLC-β isozymes for binding. Since the HTH peptide is unstructured in the absence of Gαq, we hypothesized that embedding the HTH in a folded protein might stabilize the binding-competent conformation and further improve the potency of inhibition. Using the molecular modeling software Rosetta, we searched the Protein Data Bank for proteins with similar HTH structures near their surface. The candidate proteins were computationally docked against Gαq, and their surfaces were redesigned to stabilize this interaction. We then used yeast surface display to affinity mature the designs. The most potent design bound Gαq/i with high affinity in vitro (KD = 18 nM) and inhibited activation of PLC-β isozymes in HEK293 cells. We anticipate that our genetically encoded inhibitor will help interrogate the role of Gαq in healthy and disease model systems. Our work demonstrates that grafting interaction motifs into folded proteins is a powerful approach for generating inhibitors of protein–protein interactions.  相似文献   

8.
9.
10.
Autophagy is the main lysosomal catabolic process that becomes activated under stress conditions, such as amino acid starvation and cytosolic Ca2+ upload. However, the molecular details on how both conditions control autophagy are still not fully understood. Here we link essential amino acid starvation and Ca2+ in a signaling pathway to activate autophagy. We show that withdrawal of essential amino acids leads to an increase in cytosolic Ca2+, arising from both extracellular medium and intracellular stores, which induces the activation of adenosine monophosphate-activated protein kinase (AMPK) via Ca2+/calmodulin-dependent kinase kinase-β (CaMKK-β). Furthermore, we show that autophagy induced by amino acid starvation requires AMPK, as this induction is attenuated in its absence. Subsequently, AMPK activates UNC-51-like kinase (ULK1), a mammalian autophagy-initiating kinase, through phosphorylation at Ser-555 in a process that requires CaMKK-β. Finally, the mammalian target of rapamycin complex C1 (mTORC1), a negative regulator of autophagy downstream of AMPK, is inhibited by amino acid starvation in a Ca2+-sensitive manner, and CaMKK-β appears to be important for mTORC1 inactivation, especially in the absence of extracellular Ca2+. All these results highlight that amino acid starvation regulates autophagy in part through an increase in cellular Ca2+ that activates a CaMKK-β-AMPK pathway and inhibits mTORC1, which results in ULK1 stimulation.  相似文献   

11.
Activation of T cells upon engagement of the T cell antigen receptor rapidly leads to a number of phosphorylation and plasma membrane recruitment events. For example, translocation of phospholipase-Cγ1 (PLC−γ1) to the plasma membrane and its association with the transmembrane adapter protein LAT and two other adapter proteins, Gads and SLP-76, are critical events in the early T cell activation process. We have previously characterized the formation of a tetrameric LAT-Gads-SLP-76-PLC−γ1 complex by reconstitution in vitro and have also characterized the thermodynamics of tetramer formation. In the current study, we define how PLC−γ1 recruitment to liposomes, which serve as a plasma membrane surrogate, and PLC−γ1 activation are regulated both independently and additively by recruitment of PLC−γ1 to phosphorylated LAT, by formation of the LAT-Gads-SLP-76-PLC−γ1 tetramer, and by tyrosine phosphorylation of PLC−γ1. The recently solved structure of PLC−γ1 indicates that, in the resting state, several PLC−γ1 domains inhibit its enzymatic activity and contact with the plasma membrane. We propose the multiple cooperative steps that we observed likely lead to conformational alterations in the regulatory domains of PLC−γ1, enabling contact with its membrane substrate, disinhibition of PLC−γ1 enzymatic activity, and production of the phosphoinositide cleavage products necessary for T cell activation.  相似文献   

12.
13.
Microinjection of a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa (tr-kit) activates mouse eggs parthenogenetically, and tr-kit– induced egg activation is inhibited by preincubation with an inhibitor of phospholipase C (PLC) (Sette, C., A. Bevilacqua, A. Bianchini, F. Mangia, R. Geremia, and P. Rossi. 1997. Development [Camb.]. 124:2267–2274). Co-injection of glutathione-S-transferase (GST) fusion proteins containing the src-homology (SH) domains of the γ1 isoform of PLC (PLCγ1) competitively inhibits tr-kit– induced egg activation. A GST fusion protein containing the SH3 domain of PLCγ1 inhibits egg activation as efficiently as the whole SH region, while a GST fusion protein containing the two SH2 domains is much less effective. A GST fusion protein containing the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit–induced egg activation, showing that the effect of the SH3 domain of PLCγ1 is specific. Tr-kit–induced egg activation is also suppressed by co-injection of antibodies raised against the PLCγ1 SH domains, but not against the PLCγ1 COOH-terminal region. In transfected COS cells, coexpression of PLCγ1 and tr-kit increases diacylglycerol and inositol phosphate production, and the phosphotyrosine content of PLCγ1 with respect to cells expressing PLCγ1 alone. These data indicate that tr-kit activates PLCγ1, and that the SH3 domain of PLCγ1 is essential for tr-kit–induced egg activation.  相似文献   

14.
We previously reported that the skeletal muscle-specific overexpression of Fyn in mice resulted in a severe muscle wasting phenotype despite the activation of mTORC1 signaling. To investigate the bases for the loss of muscle fiber mass, we examined the relationship between Fyn activation of mTORC1, JNK, and endoplasmic reticulum stress. Overexpression of Fyn in skeletal muscle in vivo and in HEK293T cells in culture resulted in the activation of IRE1α and JNK, leading to increased cell death. Fyn synergized with the general endoplasmic reticulum stress inducer thapsigargin, resulting in the activation of IRE1α and further accelerated cell death. Moreover, inhibition of mTORC1 with rapamycin suppressed IRE1α activation and JNK phosphorylation, resulting in protecting cells against Fyn- and thapsigargin-induced cell death. Moreover, rapamycin treatment in vivo reduced the skeletal muscle IRE1α activation in the Fyn-overexpressing transgenic mice. Together, these data demonstrate the presence of a Fyn-induced endoplasmic reticulum stress that occurred, at least in part, through the activation of mTORC1, as well as subsequent activation of the IRE1α-JNK pathway driving cell death.  相似文献   

15.
Regulator of G protein signaling (RGS) proteins interact with activated Gα subunits via their RGS domains and accelerate the hydrolysis of GTP. Although the R4 subfamily of RGS proteins generally accepts both Gαi/o and Gαq/11 subunits as substrates, the R7 and R12 subfamilies select against Gαq/11. In contrast, only one RGS protein, RGS2, is known to be selective for Gαq/11. The molecular basis for this selectivity is not clear. Previously, the crystal structure of RGS2 in complex with Gαq revealed a non-canonical interaction that could be due to interfacial differences imposed by RGS2, the Gα subunit, or both. To resolve this ambiguity, the 2.6 Å crystal structure of RGS8, an R4 subfamily member, was determined in complex with Gαq. RGS8 adopts the same pose on Gαq as it does when bound to Gαi3, indicating that the non-canonical interaction of RGS2 with Gαq is due to unique features of RGS2. Based on the RGS8-Gαq structure, residues in RGS8 that contact a unique α-helical domain loop of Gαq were converted to those typically found in R12 subfamily members, and the reverse substitutions were introduced into RGS10, an R12 subfamily member. Although these substitutions perturbed their ability to stimulate GTP hydrolysis, they did not reverse selectivity. Instead, selectivity for Gαq seems more likely determined by whether strong contacts can be maintained between α6 of the RGS domain and Switch III of Gαq, regions of high sequence and conformational diversity in both protein families.  相似文献   

16.
Sustained activation of multiple receptor tyrosine kinases (RTKs) simultaneously is vital for tumorigenesis and progression of osteosarcoma (OS). Gαi proteins recruitment to various RTKs mediates downstream oncogenic signaling activation. The expression, functions and underlying mechanisms of Gαi3 in human OS were examined. Expression of Gαi3 is robustly elevated in human OS tissues and is correlated with a poor overall survival. In patient-derived primary OS cells and immortalized lines (MG63 and U2OS), Gαi3 depletion, by shRNA and CRISPR/Cas9 strategies, robustly suppressed cell viability, proliferation and migration, while provoking G1-S arrest and apoptosis activation. Conversely, Gαi3 overexpressing ectopically can accelerate proliferation and migration of OS cells. In OS cells, Gαi3 immunoprecipitated with VEGFR2, FGFR, PGDFR and EGFR, mediating downstream cascade transduction. Akt-mTOR activation in primary OS cells was potently inhibited by Gαi3 shRNA, knockout or dominant negative mutation, but augmented after Gαi3 overexpression. In vivo studies showed that Gαi3 shRNA AAV (adeno-associated viruses) intratumoral injection largely inhibited the growth of subcutaneous xenografts of primary OS cells. Moreover, the growth of the Gαi3-knockout primary OS xenografts was much slower than that of OS xenografts with empty vector. In Gαi3-depleted OS xenografts tissues, Gαi3 downregulation and Akt-mTOR inactivation were detected. Taken together, overexpressed Gαi3 mediates RTK-Akt signaling to drive OS progression.  相似文献   

17.
CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for β-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the 15N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by GSK3bβ but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3β binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3β able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of β-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3β may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with β-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.  相似文献   

18.
In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE receptor (FcεRI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P3 production. Using immune complex phospholipase assays, we show that FcεRI cross-linking activates both PLCγ1 and PLCγ2. Activation is accompanied by the increased phosphorylation of both PLCγ isoforms on serine and tyrosine in antigen-treated cells. We also show that the two PLCγ isoforms have distinct subcellular localizations. PLCγ1 is primarily cytosolic in resting RBL-2H3 cells, with low levels of plasma membrane association. After antigen stimulation, PLCγ1 translocates to the plasma membrane where it associates preferentially with membrane ruffles. In contrast, PLCγ2 is concentrated in a perinuclear region near the Golgi and adjacent to the plasma membrane in resting cells and does not redistribute appreciably after FcεRI cross-linking. The activation of PLCγ1, but not of PLCγ2, is blocked by wortmannin, a PI 3-kinase inhibitor previously shown to block antigen-stimulated ruffling and to inhibit Ins(1,4,5)P3 synthesis. In addition, wortmannin strongly inhibits the antigen-stimulated phosphorylation of both serine and tyrosine residues on PLCγ1 with little inhibition of PLCγ2 phosphorylation. Wortmannin also blocks the antigen-stimulated translocation of PLCγ1 to the plasma membrane. Our results implicate PI 3-kinase in the phosphorylation, translocation, and activation of PLCγ1. Although less abundant than PLCγ2, activated PLCγ1 may be responsible for the bulk of antigen-stimulated Ins(1,4,5)P3 production in RBL-2H3 cells.  相似文献   

19.
The serine/threonine protein kinase YpkA is an essential virulence factor produced by pathogenic Yersinia species. YpkA is delivered into host mammalian cells via a type III secretion system and localizes to the inner side of the plasma membrane. We have previously shown that YpkA binds to and phosphorylates the α subunit of the heterotrimeric G protein complex, Gαq, resulting in inhibition of Gαq signaling. To identify residues in YpkA involved in substrate binding activity we generated GFP-YpkA N-terminal deletion mutants and performed coimmunoprecipitation experiments. We located a substrate-binding domain on amino acids 40–49 of YpkA, which lies within the previously identified membrane localization domain on YpkA. Deletion of amino acids 40–49 on YpkA interfered with substrate binding, substrate phosphorylation and substrate inhibition. Autophosphorylation regulates the kinase activity of YpkA. To dissect the mechanism by which YpkA transmits signals, we performed nano liquid chromatography coupled to tandem mass spectrometry to map in vivo phosphorylation sites. Multiple serine phosphorylation sites were identified in the secretion/translocation region, kinase domain, and C-terminal region of YpkA. Using site-directed mutagenesis we generated multiple YpkA constructs harboring specific serine to alanine point mutations. Our results demonstrate that multiple autophosphorylation sites within the N terminus regulate YpkA kinase activation, whereas mutation of serine to alanine within the C terminus of YpkA had no effect on kinase activity. YpkA autophosphorylation on multiple sites may be a strategy used by pathogenic Yersinia to prevent inactivation of this important virulence protein by host proteins.  相似文献   

20.
We showed previously that active PKC-α maintains F0F1-ATPase activity, whereas inactive PKC-α mutant (dnPKC-α) blocks recovery of F0F1-ATPase activity after injury in renal proximal tubules (RPTC). This study tested whether mitochondrial PKC-α interacts with and phosphorylates F0F1-ATPase. Wild-type PKC-α (wtPKC-α) and dnPKC-α were overexpressed in RPTC to increase their mitochondrial levels, and RPTC were exposed to oxidant or hypoxia. Mitochondrial levels of the γ-subunit, but not the α- and β-subunits, were decreased by injury, an event associated with 54% inhibition of F0F1-ATPase activity. Overexpressing wtPKC-α blocked decreases in γ-subunit levels, maintained F0F1-ATPase activity, and improved ATP levels after injury. Deletion of PKC-α decreased levels of α-, β-, and γ-subunits, decreased F0F1-ATPase activity, and hindered the recovery of ATP content after RPTC injury. Mitochondrial PKC-α co-immunoprecipitated with α-, β-, and γ-subunits of F0F1-ATPase. The association of PKC-α with these subunits decreased in injured RPTC overexpressing dnPKC-α. Immunocapture of F0F1-ATPase and immunoblotting with phospho(Ser) PKC substrate antibody identified phosphorylation of serine in the PKC consensus site on the α- or β- and γ-subunits. Overexpressing wtPKC-α increased phosphorylation and protein levels, whereas deletion of PKC-α decreased protein levels of α-, β-, and γ-subunits of F0F1-ATPase in RPTC. Phosphoproteomics revealed phosphorylation of Ser146 on the γ subunit in response to wtPKC-α overexpression. We concluded that active PKC-α 1) prevents injury-induced decreases in levels of γ subunit of F0F1-ATPase, 2) interacts with α-, β-, and γ-subunits leading to increases in their phosphorylation, and 3) promotes the recovery of F0F1-ATPase activity and ATP content after injury in RPTC.  相似文献   

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