Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Racs are involved in the regulation of important cellular processes including mitogenesis. We found that the E3 ubiquitination ligase subunit Cullin-1 interacts with constitutively active Rac3 but not with wild-type Rac3 in yeast. In mammalian cell lysates, Cullin-1 bound to V12Rac3, effector domain mutants V12L37Rac3 and V12H40Rac3, and insert domain deletion mutant V12Rac3DeltaIns(124-135). Cullin-1 also formed a clearly detectable complex with other activated Rac3-related proteins including Rac1, Rac2, Cdc42 and RhoA but not with the distantly related small GTPase Rap1. Since the proteasome is involved in cell cycle control through the programmed degradation of cell cycle proteins, the possible regulation of Rac levels during the cell cycle was examined. However, Rac was expressed at constant levels throughout the cell cycle, and a specific proteasome inhibitor had no effect on Rac protein levels. These combined results indicate that the binding of activated Rac to Cullin-1 does not affect Rac protein levels, nor does it mediate the regulation of mitogenesis by Rac. However, Rac-Cullin-1 interactions may serve to regulate other E3 ligase functions such as subcellular localization. Indeed, activated Rac3 and Cullin-1 co-localized to the perinuclear region of the cell. We also detected complex formation between Rac and the APC component CDC23. These results indicate that Rac may regulate specific proteolytic processes through directed subcellular localization of SCF or APC complexes.
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PMID:The small GTPase Rac interacts with ubiquitination complex proteins Cullin-1 and CDC23. 1144 62

The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli has been shown to activate members of the Rho family by deamidation of glutamine 63. This amino acid is essential for hydrolysis of GTP, and any substitution results in a constitutively active Rho. Activation of Rho induces the formation of stress fibers, filopodia, and membrane ruffles due to activation of RhoA, Cdc42, and Rac, respectively. Here we show that the level of endogenous Rac decreased in CNF1-treated HEK293 and HeLa cells. The amount of mRNA remained unaffected, leaving the possibility that Rac is subject to proteolytic degradation. Treatment of cells with lactacystin, an inhibitor of the 26S proteasome, protected Rac from degradation. We have previously shown that CNF1 activates the c-Jun N-terminal kinase (JNK) only transiently in HeLa cells (M. Lerm, J. Selzer, A. Hoffmeyer, U. R. Rapp, K. Aktories, and G. Schmidt, Infect. Immun. 67:496-503, 1998). Here we show that CNF1-induced JNK activation is stabilized in the presence of lactacystin. The data indicate that Rac is degraded by a proteasome-dependent pathway in CNF1-treated cells.
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PMID:Proteasomal degradation of cytotoxic necrotizing factor 1-activated rac. 1211 11

CNF1 toxin is a virulence factor produced by uropathogenic Escherichia coli. Upon cell binding and introduction into the cytosol, CNF1 deamidates glutamine 63 of RhoA (or 61 of Rac and Cdc42), rendering constitutively active these GTPases. Unexpectedly, we measured in bladder cells a transient CNF1-induced activation of Rho GTPases, maximal for Rac. Deactivation of Rac correlated with the increased susceptibility of its deamidated form to ubiquitin/proteasome-mediated degradation. Sensitivity to ubiquitylation could be generalized to other permanent-activated forms of Rac and to its sustained activation by Dbl. Degradation of the toxin-activated Rac allowed both host cell motility and efficient cell invasion by uropathogenic bacteria. CNF1 toxicity thus results from a restricted activation of Rho GTPases through hijacking the host cell proteasomal machinery.
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PMID:CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion. 1247 2

A key task for the multifunctional von Hippel-Lindau protein (pVHL) is regulation of the activity of hypoxia-inducible factor-1alpha (HIF-1alpha) by targeting it to the proteasome for degradation under normoxia. pVHL binding to HIF-1alpha is lost under low O2 tension, leading to transcription of several genes involved in the hypoxia response. However, regulation of pVHL by hypoxia remains to be investigated. We evaluated the effects of hypoxia on pVHL expression in carcinoma and endothelial cells. We showed that hypoxia stimulates pVHL levels (2.5-fold) in renal Caki-1 cells expressing wild-type VHL (VHL+/+). This upregulation was independent of VHL status, because hypoxia also increased pVHL expression in renal 786-O cells carrying mutated VHL (VHL-/-). Hypoxia did not affect pVHL expression in endothelial cells. Hypoxia-induced pVHL in Caki-1 cells was RhoA dependent, because inhibition by exotoxin C3 prevented pVHL stimulation. Furthermore, inhibition of Rho kinase by Y-27632 blocked pVHL induction by hypoxia. During normoxia, pVHL expression was also induced in cells transfected with dominant-active RhoA. Furthermore, disruption of actin organization by chemical agents or by hypoxia stimulated pVHL expression in kidney cells. On the other hand, inhibition of MAP kinases p38 and JNK, but not MAP kinase kinase (MEK1/2), reduced pVHL upregulation by 30 and 72%, respectively, during hypoxia, supporting a significant role for these signaling pathways. Expression and phosphorylation of c-Jun were stimulated in cells transfected with dominant-active RhoA. Together, these findings demonstrate that hypoxia induces pVHL expression in renal cancer cells, and this induction is mediated by RhoA-dependent pathways.
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PMID:Hypoxia upregulates von Hippel-Lindau tumor-suppressor protein through RhoA-dependent activity in renal cell carcinoma. 1458 36

Cytotoxic necrotizing factor 1 (CNF1), a virulence factor expressed by pathogenic Escherichia coli, acts on Rho-GTPases and specifically deamidates a single glutamine residue (Gln-63 in RhoA) required for GTP hydrolysis. This modification constitutively activates the effector binding function of Rho-GTPases and eventually leads to their proteasome-mediated degradation. Previous structural investigation revealed that the CNF1 active site is located in a deep and narrow pocket and that the entrance to this pocket is formed by nine loop segments. We have examined the functional importance of five of these loops (2, 6, 7, 8, and 9) by deleting them individually. We find that deletion of proximally located loops 8 and 9 in the 32 kDa catalytic domain of CNF1 (CNF1-C) nearly or completely abolishes deamidation of RhoA in vitro, identifying a potential Rho-GTPase recognition site. Deletion of loop 7 causes protein folding errors, and deletion of loop 6 has a small effect on deamidation. In contrast, deletion of loop 2 is found to increase deamidation 5-7-fold, implying that this loop rearranges in binding RhoA. None of the loop deletions or wild-type CNF1-C is able to deamidate RhoA containing Asn-63 instead of Gln-63, suggesting that the fit between the toxin and its target is highly precise. In addition, we show that the specificity constant (k(cat)/K(m)) of CNF1-C for RhoA is 825 +/- 3 M(-1) s(-1). This modest value is consistent with the confining size of the active site pocket acting to exclude nonspecific targets but also limiting reactivity toward intended targets.
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PMID:Structural elements required for deamidation of RhoA by cytotoxic necrotizing factor 1. 1459 92

We observed evolutionary conservation of canonical nuclear localization signal sequences (K(K/R)X(K/R)) in the C-terminal polybasic regions (PBRs) of some Rac and Rho isoforms. Canonical D-box sequences (RXXL), which target proteins for proteasome-mediated degradation, are also evolutionarily conserved near the PBRs of these small GTPases. We show that the Rac1 PBR (PVKKRKRK) promotes Rac1 nuclear accumulation, whereas the RhoA PBR (RRGKKKSG) keeps RhoA in the cytoplasm. A mutant Rac1 protein named Rac1 (pbrRhoA), in which the RhoA PBR replaces the Rac1 PBR, has greater cytoplasmic localization, enhanced resistance to proteasome-mediated degradation, and higher protein levels than Rac1. Mutating the D-box by substituting alanines at amino acids 174 and 177 significantly increases the protein levels of Rac1 but not Rac1(pbrRhoA). These results suggest that Rac1 (pbrRhoA) is more resistant than Rac1 to proteasome-mediated degradative pathways involving the D-box. The cytoplasmic localization of Rac1(pbrRhoA) provides the most obvious reason for its resistance to proteasome-mediated degradation, because we show that Rac1(pbrRhoA) does not greatly differ from Rac1 in its ability to stimulate membrane ruffling or to interact with SmgGDS and IQGAP1-calmodulin complexes. These findings support the model that nuclear localization signal sequences in the PBR direct Rac1 to the nucleus, where Rac1 participates in signaling pathways that ultimately target it for degradation.
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PMID:The Rac1 C-terminal polybasic region regulates the nuclear localization and protein degradation of Rac1. 1530 4

p21-activated protein kinase (PAK)-2 is a member of the PAK family of serine/threonine kinases. PAKs are activated by the p21 G-proteins Rac and Cdc42 in response to a variety of extracellular signals and act in pathways controlling cell growth, shape, motility, survival, and death. PAK-2 is unique among the PAK family members because it is also activated through proteolytic cleavage by caspase-3 or similar proteases to generate the constitutively active PAK-2p34 fragment. Activation of full-length PAK-2 by Rac or Cdc42 stimulates cell survival and protects cells from cell death, whereas caspase-activated PAK-2p34 induces a cell death response. Caspase-activated PAK-2p34 is rapidly degraded by the 26 S proteasome, but full-length PAK-2 is not. Stabilization of PAK-2p34 by preventing its polyubiquitination and degradation results in a dramatic stimulation of cell death. Although many proteins have been shown to interact with and regulate full-length PAK-2, little is known about the regulation of caspase-activated PAK-2p34. Here, we identify PS-GAP as a regulator of caspase-activated PAK-2p34. PS-GAP is a GTPase-activating protein for Cdc42 and RhoA that was originally identified by its interaction with the tyrosine kinase PYK-2. PS-GAP interacts specifically with caspase-activated PAK-2p34, but not active or inactive full-length PAK-2, through a region between the GAP and SH3 domains. The interaction with PS-GAP inhibits the protein kinase activity of PAK-2p34 and changes the localization of PAK-2p34 from the nucleus to the perinuclear region. Furthermore, PS-GAP decreases the stimulation of cell death induced by stabilization of PAK-2p34.
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PMID:Identification and characterization of PS-GAP as a novel regulator of caspase-activated PAK-2. 1547 51

Serum response factor (SRF) is activated by contractile and hypertrophic agonists, such as endothelin-1 (ET1) to stimulate expression of cytoskeletal proteins in vascular smooth muscle cells (VSMCs). While studying the regulation of smooth muscle alpha-actin (SMA) expression at the level of protein stability, we discovered that inhibition of proteasome-dependent protein degradation by N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) or lactacystin (LC) did not enhance the levels of SMA, but, unexpectedly, attenuated SMA expression in response to ET1, without affecting the viability of VSMCs. Down-regulation of SMA protein by MG132 or LC occurred at the level of SMA transcription and via the inhibition of SRF activity. By contrast, MG132 and LC potentiated the activity of activator protein-1 transcription factor. Regulation of SRF by MG132 was not related to inhibition of nuclear factor-kappaB, an established target of proteasome inhibitors, and was not mediated by protein kinase A, a powerful regulator of SRF activity. Signaling studies indicate that inhibition of ET1-induced SRF activity by MG132 occurs at the level downstream of heterotrimeric G proteins Gq/11 and G13, of small GTPase RhoA, and of actin dynamics but at the level of SRF-DNA binding. MG132 treatment did not result in ubiquitination or accumulation of SRF. By contrast, the levels of c-Jun were rapidly increased upon incubation of cells with MG132, and ectopic overexpression of c-Jun mimicked the effect of MG132 on SRF activity. Together, these data suggest that inhibition of proteasome results in down-regulation of SMA expression via up-regulation of c-Jun and repression of SRF activity at the level of DNA binding.
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PMID:Regulation of serum response factor-dependent gene expression by proteasome inhibitors. 1555 Jun 77

cAMP and cyclic GMP-dependent kinases (PKA and PKG) phosphorylate the small G protein RhoA on Ser188. We have previously demonstrated that phosphorylation of Ser188 inhibits RhoA-dependent functions and positively regulates RhoA expression, and that the nitric oxide (NO)/cGMP-dependent protein kinase pathway plays an essential role, both in vitro and in vivo, in the regulation of RhoA protein expression and functions in vascular smooth muscle cells. Here we analyze the consequences of Ser188 phosphorylation on RhoA protein degradation. By expressing Ser188 phosphomimetic wild-type (WT-RhoA-S188E) and active RhoA proteins (Q63L-RhoA-S188E), we show that phosphorylation of Ser188 of RhoA protects RhoA, particularly its active form, from ubiquitin-mediated proteasomal degradation. Coimmunoprecipitation experiments indicate that the resistance of the phosphorylated active form of RhoA to proteasome-mediated degradation is because of its cytoplasmic sequestration through enhanced RhoGDI interaction. In rat aortic smooth muscle cells, stimulation of PKG and inhibition of proteasome by lactacystin, induce nonadditive increases in RhoA protein expression. In addition, stimulation of PKG leads to the accumulation of GTP-bound RhoA in the cytoplasm. In vivo stimulation of the NO/PKG signaling by treating rats with sildenafil increased RhoA level and RhoA phosphorylation, and enhanced its association to RhoGDI in the pulmonary artery, whereas opposite effects are induced by chronic inhibition of NO synthesis in N-omega-nitro-L-arginine-treated rats. Our results thus suggest that Ser188 phosphorylation-mediated protection against degradation is a physiological process regulating the level of endogenous RhoA and define a novel function for RhoGDI, as an inhibitor of Rho protein degradation.
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PMID:Phosphorylation of serine 188 protects RhoA from ubiquitin/proteasome-mediated degradation in vascular smooth muscle cells. 1589 Sep 75

The proto-oncogene Ras GTPase stimulates transcription of p21Waf1/Cip1 (p21), which is repressed by the RhoA GTPase. We previously showed that Ras also elevates p21 protein levels by reducing its proteasome-mediated degradation. Therefore, we investigated whether RhoA also influenced p21 protein degradation. Pulse-chase analysis of p21 protein stability revealed that inhibitors of Rho function, which disrupt filamentous actin (F-actin), drastically slowed p21 degradation. Direct F-actin disruption mimicked Rho inhibition to stabilize p21. We found that Rho inhibition, or F-actin disruption, activated the JNK stress-activated protein kinase, and that interfering with JNK signalling, but not p38, abrogated p21 stabilization by Rho inhibition or F-actin-disrupting drugs. In addition, Ras-transformation led to increased constitutive JNK activity that contributed to the elevated p21 protein levels. These data suggest that p21 stability is influenced by a mechanism that monitors F-actin downstream of Rho, and which acts through a pathway involving activation of JNK. These results may have significant implications for therapies that target Rho-signalling pathways to induce p21-mediated cell-cycle arrest.
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PMID:Stability of p21Waf1/Cip1 CDK inhibitor protein is responsive to RhoA-mediated regulation of the actin cytoskeleton. 1640 39


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