p50<sup><i>cdc37</i></sup> Acting in Concert with Hsp90 Is Required for Raf-1 Function

Grammatikakis, Nicholas; Lin, Jun-Hsiang; Grammatikakis, Aliki; Tsichlis, Philip N.; Cochran, Brent

doi:10.1128/mcb.19.3.1661

Cited by 251 publications

(319 citation statements)

References 81 publications

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“…p50 is a kinase specific co-chaperone and is thought to assist in the transfer of kinases to Hsp90 (Grammatikakis et al, 1999;Lee et al, 2002;Stepanova et al, 1996). Domain mapping has shown that p50's N-terminal domain is critical for binding kinases whereas the middle and C-terminal domains interact with Hsp90 (Grammatikakis et al, 1999;Roe et al, 2004;Shao et al, 2003).…”

Section: P50 a Kinase Specific Co-chaperone Also Traps Hsp90 In An mentioning

confidence: 99%

Conformational dynamics of the molecular chaperone Hsp90

Krukenberg

Street

Lavery

et al. 2011

Quart. Rev. Biophys.

278

262

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The molecular chaperone Hsp90 is an essential eukaryotic protein that makes up 1-2% of all cytosolic proteins. Hsp90 is vital for the maturation and maintenance of a wide variety of substrate proteins largely involved in signaling and regulatory processes. Many of these substrates have also been implicated in cancer and other diseases making Hsp90 an attractive target for therapeutics. Hsp90 is a highly dynamic and flexible molecule that can adapt its conformation to the wide variety of substrate proteins with which it acts. Large conformational rearrangements are also required for the activation of these client proteins. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shifts the equilibrium between a pre-existing set of conformational states in an organismdependent manner. In vivo Hsp90 functions as part of larger heterocomplexes. The binding partners of Hsp90, co-chaperones, assist in the recruitment and activation of substrates, and many co-chaperones further regulate the conformational dynamics of Hsp90 by shifting the conformational equilibrium towards a particular state. Studies have also suggested alternative mechanisms for the regulation of Hsp90's conformation. In this review, we discuss the structural and biochemical studies leading to our current understanding of the conformational dynamics of Hsp90 and the role that nucleotide, co-chaperones, post-translational modification and clients play in regulating Hsp90's conformation. We also discuss the effects of current Hsp90 inhibitors on conformation and the potential for developing small molecules that inhibit Hsp90 by disrupting the conformational dynamics.

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Section: P50 a Kinase Specific Co-chaperone Also Traps Hsp90 In An mentioning

confidence: 99%

Conformational dynamics of the molecular chaperone Hsp90

Krukenberg

Street

Lavery

et al. 2011

Quart. Rev. Biophys.

278

262

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“…A possible explanation for the contrasting differential between B-Raf wt and B-Raf V600E in our study and that of Wan et al (2004) is that we used RIPA buffer for our immunoprecipitations, whereas the latter utilized more mild conditions (0.5% NP-40). Whereas our conditions are probably more specific for B-Raf (by reducing the risk of contamination with other MEK kinases such as Raf-1 ), they may lead to the dissociation of important cofactors such as the HSP90/p50cdc37 complex (Grammatikakis et al, 1999;Grbovic et al, 2006). N-region charge and Ras-GTP binding are dispensable for the transforming potential of B-Raf V600E We next analysed the potential of B-Raf V600E proteins with a neutralized N-region or a defective RBD to induce oncogenic transformation.…”

Section: Regulation Of B-raf Signalling T Brummer Et Almentioning

confidence: 99%

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

et al. 2006

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The BRAF V600E mutation is found in approximately 6% of human cancers and mimics the phosphorylation of the kinase domain activation segment. In wild-type B-Raf (B-Raf wt ), activation segment phosphorylation is thought to cooperate with negative charges within the N-region for full activation. In contrast to Raf-1, the N-region of B-Raf is constitutively negatively charged owing to the presence of residues D447/D448 and the phosphorylation of S446. Therefore, it has been suggested that this hallmark predisposes B-Raf for oncogenic activation. In this study, we demonstrate that neutralizing mutations of these residues (in particular S446 and S447), or uncoupling of B-Raf from Ras-guanine 5 0 -triphosphate (GTP), strongly reduce the biological activity of B-Raf in a PC12 cell differentiation assay. We also confirm that S365 is a 14-3-3 binding site, and determine that mutation of this residue rescues the impaired biological activity of B-Raf proteins with a neutralized N-region, suggesting that the N-region opposes a 14-3-3-mediated transition into an inactive conformation. However, in the case of B-Raf V600E , although complete N-region neutralization resulted in a 2.5-fold reduction in kinase activity in vitro, this oncoprotein strongly induced PC12 differentiation or transformation and epithelial-mesenchymal transition of MCF-10A cells regardless of its N-region charge. Furthermore, the biological activity of B-Raf V600E was independent of its ability to bind Ras-GTP. Our analysis identifies important regulatory differences between B-Raf wt and B-Raf V600E and suggests that B-Raf V600E cannot be inhibited by strategies aimed at blocking S446 phosphorylation or Ras activation. Keywords: Ras; 14-3-3 proteins; b-Catenin; E-cadherin; mammary epithelial cells IntroductionThe Ras/Raf/mitogen-activated/extracellular-regulated kinase (MEK)/extracellular signal regulated kinase (ERK) pathway plays a pivotal role in control of proliferation and differentiation and, owing to its role as a gatekeeper of this pathway, Raf appears an attractive therapeutic target (O'Neill and Kolch, 2004;Wilhelm et al., 2004). The Raf-kinase family comprises the A-Raf, B-Raf and Raf-1 isoforms in vertebrates as well as D-Raf and LIN-45 in Drosophila and Caenorhabditis, respectively. B-Raf, the major ERK activator in vertebrates, is required for the maintenance of basal ERK activity and displays the most potent transforming activity (Papin et al., 1998;Brummer et al., 2002;Mercer and Pritchard, 2003). All isoforms share three highly conserved regions (CRs; Figure 1a): the N-terminal CR1 contains the Ras-guanine 5 0 -triphosphate (GTP)-binding domain (RBD), which initiates the interaction with Ras-GTP through a conserved arginine residue (R188 in B-Raf) that is required for the recruitment and activation of Raf at the plasma membrane. Consequently, mutation of this residue prevents Ras/Raf interaction and renders D-Raf, B-Raf and Raf-1 unresponsive to most extracellular signals (Hou et al., 1995;Marais et al., 1997;Brummer et al., 2002). The ...

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“…A number of chaperones have been found to associate with Raf-1, including Hsp90 (heat-shock protein of 90 kDa) and Hsp50\ Cdc37 [16,95,96], FKBP65 (FK-506 binding protein) [97] and Bag-1 [98]. Bag-1 was isolated originally as an anti-apoptotic Bcl-2 binding partner, but subsequent experimentation has placed it among the chaperones by revealing that it regulates Hsp70 and Hsc70 [99,100].…”

Section: Staying In Shape and More : What Chaperones Do For Rafmentioning

confidence: 99%

“…A first hint was provided by the observation that, in PC12 cells, the preferential activation of B-Raf over Raf-1 was correlated with Hsp90 association [105]. Later on it was shown that the association of Raf-1 with Hsp90 was primarily mediated via Cdc37 [95,96]. The overexpression of a Cdc37 mutant deficient in Hsp90 binding impaired the growthfactor stimulation of the ERK pathway in mammalian cells, whereas the co-expression of Cdc37 in insect cells enhanced both the basal and the Ras-and Src-induced activity of Raf-1.…”

Section: Staying In Shape and More : What Chaperones Do For Rafmentioning

confidence: 99%

“…Cdc37 could even partially restore the activity of a Raf-1 serine-621 mutant, suggesting functional resemblance to 14-3-3. In contrast, a Raf-1 mutant in which the tyrosine phosphorylation site had been replaced was resistant to Cdc37 activation [96]. As the Cdc37-Hsp90 complex is known to associate with v-Src and other tyrosine kinases [106,107], an intriguing possibility is that Cdc37 links Raf-1 to activation by tyrosine phosphorylation.…”

Section: Staying In Shape and More : What Chaperones Do For Rafmentioning

confidence: 99%

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Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions

Kölch

2000

Biochemical Journal

1,046

118

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The Ras\Raf\MEK (mitogen-activated protein kinase\ERK kinase)\ERK (extracellular-signal-regulated kinase) pathway is at the heart of signalling networks that govern proliferation, differentiation and cell survival. Although the basic regulatory steps have been elucidated, many features of this pathway are only beginning to emerge. This review focuses on the role of protein-protein interactions in the regulation of this pathway, INTRODUCTIONThe mitogen-activated protein kinase (MAPK) pathway is one of the primordial signalling systems that Nature has used in several permutations to accomplish an amazing variety of tasks. It exists in all eukaryotes, and controls such fundamental cellular processes as proliferation, differentiation, survival and apoptosis. The basic arrangement includes a G-protein working upstream of a core module consisting of three kinases : a MAPK kinase kinase (MAPKKK) that phosphorylates and activates a MAPK kinase (MAPKK), which in turn activates MAPK ( Figure 1). This set-up provides not only for signal amplification, but, maybe even more importantly, for additional regulatory interfaces that allow the kinetics, duration and amplitude of the activity to be precisely tuned. At present we can distinguish six MAPK modules, which share structurally related components, but seem to mediate specific biological responses. For recent reviews on MAPK pathways, the reader is referred to references [1][2][3]. Here we will focus on the regulation of the ERK (extracellular-signal-regulated kinase) pathway, which features Ras as G-protein, Raf as MAPKKK, MEK (MAPK\ERK kinase) as MAPKK and ERK as MAPK.Despite enjoying a decade in the limelight of scientific interest and revealing a plethora of new insights into the circuitry of signalling pathways in general, this pathway still holds many secrets. These pertain mainly to the regulation of Raf, and to a deeper understanding of how specific biological responses are encoded by spatial and temporal changes in the activity and subcellular distribution of the pathway components, and how these fluctuations are orchestrated at the molecular level. Work from many laboratories has highlighted protein-protein interactions as powerful means of co-ordinating signalling processes, most strikingly exemplified by the assembly of multi-protein signalling complexes on activated receptors, or of transcriptionfactor complexes on gene promoters. However, it is becoming Abbreviations used : Bcr, Breakpoint cluster region ; BXB, isolated Raf-1 kinase domain ; CNK, connector-enhancer of KSR ; CK2, casein kinase 2 ; CRD, cysteine-rich domain ; DEF, docking site for ERK, FxFP ; DLK, dual leucine-zipper-bearing kinase ; ERK, extracellular-signal-regulated kinase ; Hsp, heat-shock protein ; KIM, kinase interaction motif ; IκB, inhibitor of NF-κB ; JNK, c-Jun N-terminal kinase ; KSR, kinase suppressor of Ras ; MAPK, mitogen-activated protein kinase ; MAPKK, MAPK kinase ; MAPKKK, MAPK kinase kinase ; MEK, MAPK/ERK kinase ; MEKK, MEK kinase ; MP1, MEK partner 1 ; MUK, MAPK upstream kin...

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p50^cdc37 Acting in Concert with Hsp90 Is Required for Raf-1 Function

Cited by 251 publications

References 81 publications

Conformational dynamics of the molecular chaperone Hsp90

Conformational dynamics of the molecular chaperone Hsp90

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions

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p50cdc37 Acting in Concert with Hsp90 Is Required for Raf-1 Function

Cited by 251 publications

References 81 publications

Conformational dynamics of the molecular chaperone Hsp90

Conformational dynamics of the molecular chaperone Hsp90

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions

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p50^cdc37 Acting in Concert with Hsp90 Is Required for Raf-1 Function