Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cellular N-Ras provides a steady-state antiapoptotic signal, at least partially through the regulation of phosphorylated Akt and Bad levels. Fibroblasts lacking c-N-Ras expression are highly sensitive to the induction of apoptosis by a variety of agents. Reduction of pBad and pAkt levels using a phosphatidylinositol 3-kinase inhibitor was not sufficient to sensitize the control cell population to the high level of apoptosis observed in the N-Ras knockout cell lines, suggesting that c-N-Ras provides at least one other antiapoptotic signal. Stimulation of the control cells with apoptotic agents results in a transient increase in Jun N-terminal protein kinase (JNK)/p38 activity that decreased to baseline levels during the time course of the experiments. In all cases, however, sustained JNK/p38 activity was observed in cells lacking c-N-Ras expression. This correlated with sustained levels of phosphorylated MKK4 and MKK3/6, upstream activators of JNK and p38, respectively. Mimicking the sustained activation of JNK in the control cells did result in increasing their sensitivity to apoptotic agents, suggesting that prolonged JNK activity is a proapoptotic event. We also examined the potential downstream c-N-Ras targets that might be involved in regulating the duration of the JNK/p38 signal. Only the RalGDS 37G-N-Ras protein protected the N-Ras knockout cells from apoptosis and restored transient rather than sustained JNK activation. These data suggest that cellular N-Ras provides an antiapoptotic signal through at least two distinct mechanisms, one which regulates steady-state pBad and pAkt levels and one which regulates the duration of JNK/p38 activity following an apoptotic challenge.
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PMID:Cellular N-Ras promotes cell survival by downregulation of Jun N-terminal protein kinase and p38. 1183 24

Thyrotropin (TSH) is considered the main regulator of thyrocyte differentiation and proliferation. Thus, the characterization of the different signaling pathways triggered by TSH on these cells is of major interest in order to understand the mechanisms implicated in thyroid pathology. In this review we focus on the different signaling pathways involved in TSH-mediated proliferation and their role in thyroid transformation and tumorigenesis. TSH mitogenic activities are mediated largely by cAMP, which in turn may activate protein kinase (PKA)-dependent and independent processes. We analyze the effects of increased cAMP levels and PKA activity during cell cycle progression and the role of this signaling pathway in thyroid tumor initiation. Alternative pathways to PKA in the cAMP-mediated proliferation appear to involve the small GTPases Rap1 and Ras. We analyze the Ras effectors (PI3K, RalGDS and Raf) that are thought to mediate its oncogenic activity, as well as the ability of Ras to induce apoptosis in thyrocytes. Finally, we discuss the activation of the PLC/PKC cascade by TSH in thyroid cells and the role of this signaling pathway in the TSH-mediated proliferation and tumorigenesis.
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PMID:TSH-activated signaling pathways in thyroid tumorigenesis. 1506 72

The guanine nucleotide-binding protein Ras occurs in solution in two different states, state 1 and state 2, when the GTP analogue GppNHp is bound to the active center as detected by (31)P NMR spectroscopy. Here we show that Ras(wt).Mg(2+).GppCH(2)p also exists in two conformational states in dynamic equilibrium. The activation enthalpy DeltaH(++)(12) and the activation entropy DeltaS(++)(12) for the transition from state 1 to state 2 are 70 kJ mol(-1) and 102 J mol(-1) K(-1), within the limits of error identical to those determined for the Ras(wt).Mg(2+).GppNHp complex. The same is true for the equilibrium constants K(12) = [2]/[1] of 2.0 and the corresponding DeltaG(12) of -1.7 kJ mol(-1) at 278 K. This excludes a suggested specific effect of the NH group of GppNHp on the equilibrium. The assignment of the phosphorus resonance lines of the bound analogues has been done by two-dimensional (31)P-(31)P NOESY experiments which lead to a correction of the already reported assignments of bound GppNHp. Mutation of Thr35 in Ras.Mg(2+).GppCH(2)p to serine leads to a shift of the conformational equilibrium toward state 1. Interaction of the Ras binding domain (RBD) of Raf kinase or RalGDS with Ras(wt) or Ras(T35S) shifts the equilibrium completely to state 2. The (31)P NMR experiments suggest that, besides the type of the side chain of residue 35, a main contribution to the conformational equilibrium in Ras complexes with GTP and GTP analogues is the effective acidity of the gamma-phosphate group of the bound nucleotide. A reaction scheme for the Ras-effector interaction is presented which includes the existence of two conformations of the effector loop and a weak binding state.
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PMID:Conformational states of Ras complexed with the GTP analogue GppNHp or GppCH2p: implications for the interaction with effector proteins. 1569 48

Liquid-state 31P NMR spectroscopy is a well-established method for the study of guanine nucleotide-binding proteins (GNB proteins) such as the proto-oncogene Ras. Solid-state 31P NMR spectroscopy could meanwhile also be used to study microcrystalline samples of Ras as well as its partial loss-of-function mutants Ras(T35S) and Ras(T35A). However, solid-state NMR studies of the latter mutants in complex with effector molecules such as RalGDS or Raf kinase were so far prevented, since it has been impossible to crystallize these complexes yet. The aim of the present contribution is to make such complexes accessible to solid-state 31P NMR spectroscopy by the application of precipitation methods. The complex formed by Ras(T35S) and Raf kinase is preserved during precipitation. In contrast, the weakly bound complex of Ras(T35S) with RalGDS is dissociated or at least perturbed by the precipitation procedure. Solid-state 31P NMR experiments on precipitates of these complexes deliver spectra of high resolution and signal-to-noise ratio which allows the application of two-dimensional techniques. Precipitates prepared using polyethylene glycol 6000 (PEG) as precipitant were found to exhibit spectra of maximum resolution and signal-to-noise ratio. Interestingly, the 31P signal due to the alpha-phosphate of GppNHp bound to Ras(T35S) in crystalline samples or aged precipitates has a significantly different isotropic chemical shift than in the liquid state or in freshly prepared precipitates. This directly indicates that the crystal structure differs from the equilibrium solution structure at least in the neighborhood of the alpha-phosphate group.
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PMID:Solid-state 31P NMR spectroscopy of precipitated guanine nucleotide-binding protein Ras in complexes with its effector molecules Raf kinase and RalGDS. 1731 21

The neurofibromatosis 2 (NF2) tumor suppressor protein merlin is commonly mutated in human benign brain tumors. The gene altered in NF2 was located on human chromosome 22q12 in 1993 and the encoded protein named merlin and schwannomin. Merlin has homology to ERM family proteins, ezrin, radixin, and moesin, within the protein 4.1 superfamily. In efforts to determine merlin function several groups have discovered 34 merlin interacting proteins, including ezrin, radixin, moesin, CD44, layilin, paxillin, actin, N-WASP, betaII-spectrin, microtubules, TRBP, eIF3c, PIKE, NHERF, MAP, RalGDS, RhoGDI, EG1/magicin, HEI10, HRS, syntenin, caspr/paranodin, DCC, NGB, CRM1/exportin, SCHIP1, MYPT-1-PP1delta, RIbeta, PKA, PAK (three types), calpain and Drosophila expanded. Many of the proteins that interact with the merlin N-terminal domain also bind ezrin, while other merlin interacting proteins do not bind other members of the ERM family. Merlin also interacts with itself. This review describes these proteins, their possible roles in NF2, and the resultant hypothesized merlin functions. Review of all of the merlin interacting proteins and functional consequences of losses of these interactions reveals multiple merlin actions in PI3-kinase, MAP kinase and small GTPase signaling pathways that might be targeted to inhibit the proliferation of NF2 tumors.
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PMID:The merlin interacting proteins reveal multiple targets for NF2 therapy. 1798 Jan 64

RGL2 [RalGDS (Ral guanine nucleotide dissociation stimulator)-like 2] is a member of the RalGDS family that we have previously isolated and characterized as a potential effector for Ras and the Ras analogue Rap1b. The protein shares 89% sequence identity with its mouse orthologue Rlf (RalGDS-like factor). In the present study we further characterized the G-protein-binding features of RGL2 and also demonstrated that RGL2 has guanine-nucleotide-exchange activity toward the small GTPase RalA. We found that RGL2/Rlf properties are well conserved between human and mouse species. Both RGL2 and Rlf have a putative PKA (protein kinase A) phosphorylation site at the C-terminal of the domain that regulates the interaction with small GTPases. We demonstrated that RGL2 is phosphorylated by PKA and phosphorylation reduces the ability of RGL2 to bind H-Ras. As RGL2 and Rlf are unique in the RalGDS family in having a PKA site in the Ras-binding domain, the results of the present study indicate that Ras may distinguish between the different RalGDS family members by their phosphorylation by PKA.
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PMID:G-protein binding features and regulation of the RalGDS family member, RGL2. 1854 Aug 61

The GTP-binding protein Ras plays a central role in the regulation of various cellular processes, acting as a molecular switch that triggers signaling cascades. Only Ras bound to GTP is able to interact strongly with effector proteins like Raf kinase, phosphatidylinositol 3-kinase, and RalGDS, whereas in the GDP-bound state, the stability of the complex is strongly decreased, and signaling is interrupted. To determine whether this process is only controlled by the stability of the complex, we used computer-aided protein design to improve the interaction between Ras and effector. We challenged the Ras.Raf complex in this study because Raf among all effectors shows the highest Ras affinity and the fastest association kinetics. The proposed mutations were characterized as to their changes in dynamics and binding strength. We demonstrate that Ras-Raf interaction can only be improved at the cost of a loss in specificity of Ras.GTP versus Ras.GDP. As shown by NMR spectroscopy, the Raf mutation A85K leads to a shift of Ras switch I in the GTP-bound as well as in the GDP-bound state, thereby increasing the complex stability. In a luciferase-based reporter gene assay, Raf A85K is associated with higher signaling activity, which appears to be a mere matter of Ras-Raf affinity.
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PMID:Improved binding of raf to Ras.GDP is correlated with biological activity. 1977 12

Mutant K-Ras and survivin both contribute to oncogenesis, but little is known about K-Ras requirement for the maintenance of the high levels of survivin in human tumors. Here we demonstrate that K-Ras depletion significantly decreases survivin levels in human cancer cells that harbor mutant but not wild type K-Ras. K-Ras depletion attenuates both basal and drug-induced survivin levels. The mechanism by which K-Ras depletion decreases survivin levels is through ubiquitination and proteasomal degradation of survivin and is independent of survivin-Thr-34 phosphorylation. Depletion of RalA and RalB, but not Raf-1, Akt1 and Akt2, decreases survivin levels, suggesting that K-Ras may regulate survivin stability through its RalGDS/Ral but not PI3K/Akt and Raf-1/Mek effector pathways. Furthermore, the ability of mutant K-Ras to induce anchorage-independent growth, invasion and survival is compromised by depletion of survivin. These studies suggest that mutant K-Ras contributes to the maintenance of the aberrantly high levels of survivin in tumors by regulating its stability, and that the ability of mutant K-Ras to induce malignant transformation is, at least in part, dependent on these high levels of survivin.
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PMID:Depletion of K-Ras promotes proteasome degradation of survivin. 2332 41


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