Phosphorylation Analysis of 90 kDa Heat Shock Protein within the Cytosolic Arylhydrocarbon Receptor Complex

Ogiso, Hideo; Kagi, Noriko; Matsumoto, Emiko; Nishimoto, Madoka; Arai, Ryoichi; Shirouzu, Mikako; Mimura, Junsei; Fujii‐Kuriyama, Yoshiaki; Yokoyama, Shigeyuki

doi:10.1021/bi048736m

Cited by 77 publications

(67 citation statements)

References 70 publications

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“…Hyperphosphorylation at these sites has been shown to result in the inhibition of the chaperoning function of Hsp90 (Mimnaugh et al, 1995;Zhao et al, 2001;Ogiso et al, 2004). Similarly, our results suggest that, under basal conditions, Y300 phosphorylation could reduce the chaperoning role of Hsp90 with client proteins ( Figure 6E).…”

Section: Discussionsupporting

confidence: 80%

“…Hyperphosphorylation of serine and/or threonine residues on Hsp90 has been shown to negatively regulate Hsp90's role in the conformational maturation of oncogenic signaling proteins, including ErbB2, c-Src, Akt, and Raf-1 (Schulte et al, 1995;Mimnaugh et al, 1995;Xu et al, 2001;Zhao et al, 2001;Basso et al, 2002). However, details in the understanding of the role of these phosphorylation sites in Hsp90 function are still emerging (Ogiso et al, 2004). Furthermore, evidences for a role of regulated posttranslational modifications in intracellular signaling events are lacking.…”

Section: Introductionmentioning

confidence: 99%

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Src-mediated Phosphorylation of Hsp90 in Response to Vascular Endothelial Growth Factor (VEGF) Is Required for VEGF Receptor-2 Signaling to Endothelial NO Synthase

Duval

Bœuf

Huot

et al. 2007

MBoC

130

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Nitric oxide (NO) release from endothelial cells, via endothelial NO synthase (eNOS) activation, is central to the proangiogenic actions of vascular endothelial growth factor (VEGF). VEGF signaling to eNOS is principally mediated byan Akt-dependent phosphorylation of eNOS and by increased association of eNOS to the molecular chaperone, heatshock protein 90 kDa (Hsp90). Herein, we report that VEGFR-2 activation induces tyrosine phosphorylation of VEGF receptor 2 (VEGFR-2)-associated Hsp90␤. Tyrosine phosphorylation of Hsp90␤ in response to VEGF is dependent on internalization of the VEGFR-2 and on Src kinase activation. Furthermore, we demonstrate that c-Src directly phosphorylates Hsp90 on tyrosine 300 residue and that this event is essential for VEGF-stimulated eNOS association to Hsp90 and thus NO release from endothelial cells. Our work identifies Y300 phosphorylation of Hsp90 as a novel regulated posttranslational modification of the chaperone and demonstrates its importance in the proangiogenic actions of VEGF, namely by regulating NO release from endothelial cells. INTRODUCTIONVascular endothelial growth factor (VEGF) is a potent proangiogenic cytokine that activates three specific receptor tyrosine kinases (RTK): VEGF receptor (VEGFR)-1, -2, and -3. Gene knockout studies revealed that VEGF and VEGFRs are essential for the development of the vasculature in mouse embryos (Fong et al., 1995;Shalaby et al., 1995;Carmeliet et al., 1996;Dumont et al., 1998). In addition to its functional roles in vasculogenesis, VEGF plays key roles in adult physiological and pathological angiogenesis such as wound healing and tumor vascularization. VEGFR-2 mediates most of the functional and biochemical effects of VEGF in endothelial cells including proliferation, migration, survival, and nitric oxide (NO) release. These effects are produced through the activation of multiple signaling pathways, such as the phosphoinositide-3 kinase (PI-3 kinase)/ Akt, p38 mitogen-activated protein kinase (MAPK), phospholipase C (PLC)-␥, and Erk/MAPK, after the activation of VEGFR-2 (Rousseau et al., 1997;Takahashi and Shibuya, 1997;Gerber et al., 1998;Takahashi et al., 2001). VEGF-mediated activation of the Ser/Thr kinase Akt leads to phosphorylation of serine 1179 on bovine endothelial NO synthase (eNOS; Ser 1177 in the human form), which increases eNOS catalytic activity and results in release of NO from endothelial cells (Fulton et al., 1999;Dimmeler et al., 1999). In turn, NO released from endothelium contributes to the proangiogenic effects of VEGF. Indeed, VEGF-mediated vascular permeability, angiogenesis, and endothelial cell precursor mobilization are markedly impaired in eNOS-deficient mice (Fukumura et al., 2001;Aicher et al., 2003).Heat-shock protein 90 kDa (Hsp90) is a multifunctional molecular chaperone, and its role in the prevention of protein aggregation and in the refolding of denatured proteins has been studied in much detail (Whitesell and Lindquist, 2005). Hsp90 is also a regulator of the activity of signaling molecules su...

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Section: Discussionsupporting

confidence: 80%

Section: Introductionmentioning

confidence: 99%

Src-mediated Phosphorylation of Hsp90 in Response to Vascular Endothelial Growth Factor (VEGF) Is Required for VEGF Receptor-2 Signaling to Endothelial NO Synthase

Duval

Bœuf

Huot

et al. 2007

MBoC

130

View full text Add to dashboard Cite

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“…The authors suggest that the unphosphorylated HSP90β isoform has a high affinity for the AhR and subsequently higher transcriptional activity. 150 In yeast, it appears that the AhR may preferentially utilize the constitutive HSP90β isoform over the more inducible HSP90α chaperone. 151 Further studies need to be performed to corroboratethe importance of these findings.…”

Section: Kda Heat Shock Proteinmentioning

confidence: 99%

The Aryl Hydrocarbon Receptor Complex and the Control of Gene Expression

Beischlag

Morales

Hollingshead

et al. 2008

Crit Rev Eukar Gene Expr

632

587

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The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that controls the expression of a diverse set of genes. The toxicity of the potent AhR ligand 2,3,7,8-tetrachlorodibenzop-dioxin is almost exclusively mediated through this receptor. However, the key alterations in gene expression that mediate toxicity are poorly understood. It has been established through characterization of AhR-null mice that the AhR has a required physiological function, yet how endogenous mediators regulate this orphan receptor remains to be established. A picture as to how the AhR/ARNT heterodimer actually mediates gene transcription is starting to emerge. The AhR/ ARNT complex can alter transcription both by binding to its cognate response element and through tethering to other transcription factors. In addition, many of the coregulatory proteins necessary for AhR-mediated transcription have been identified. Cross talk between the estrogen receptor and the AhR at the promoter of target genes appears to be an important mode of regulation. Inflammatory signaling pathways and the AhR also appear to be another important site of cross talk at the level of transcription. A major focus of this review is to highlight experimental efforts to characterize nonclassical mechanisms of AhR-mediated modulation of gene transcription.

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“…As with acetylation, increased phosphorylation negatively regulates Hsp90's interaction with client proteins (Adinolfi et al, 2003;Ogiso et al, 2004;Wandinger et al, 2006). The phosphatase PP5 directly associates with Hsp90 via a TPRdomain, and PP5 directly dephosphorylates Hsp90 .…”

Section: The Effect Of Phosphorylation and Acetylation On The Conformmentioning

confidence: 99%

“…Evidence suggests that phosphorylation may be more complex with differing roles on Hsp90 function depending on the client protein involved. Phosphorylation at S225 and S254 in Hsp90 causes a decreased affinity for the aryl hydrocarbon receptor (AhR) (Ogiso et al, 2004). In another example, c-src directly phosphorylates Hsp90 on Y300 and unlike other clients this phosphorylation event is required for the binding of eNOS to Hsp90 (Duval et al, 2007).…”

Section: The Effect Of Phosphorylation and Acetylation On The Conformmentioning

confidence: 99%

Conformational dynamics of the molecular chaperone Hsp90

Krukenberg

Street

Lavery

et al. 2011

Quart. Rev. Biophys.

271

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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Phosphorylation Analysis of 90 kDa Heat Shock Protein within the Cytosolic Arylhydrocarbon Receptor Complex

Cited by 77 publications

References 70 publications

Src-mediated Phosphorylation of Hsp90 in Response to Vascular Endothelial Growth Factor (VEGF) Is Required for VEGF Receptor-2 Signaling to Endothelial NO Synthase

Src-mediated Phosphorylation of Hsp90 in Response to Vascular Endothelial Growth Factor (VEGF) Is Required for VEGF Receptor-2 Signaling to Endothelial NO Synthase

The Aryl Hydrocarbon Receptor Complex and the Control of Gene Expression

Conformational dynamics of the molecular chaperone Hsp90

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