Temperature-sensitive ZAP70 mutants degrading through a proteasome-independent pathway. Restoration of a kinase domain mutant by Cdc37

CD8 deficiency is an autosomal recessive form of severe combined immunodeficiency diseases characterized by the absence of CD8(+) T lymphocytes and impaired T cell functions. We identified two novel mis-sense mutations in the zap70 genes of a CD8-deficiency patient. One mutation (P80Q) affects a residue in an SH2 domain and another (M572L) in the kinase subdomain XI. Both mutations cause a degradation of ZAP70 protein in a temperature-sensitive manner through an ATP-dependent and proteasome-independent pathway. We further demonstrated that Cdc37, a protein kinase-specific chaperone, bound to M572L but not P80Q mutant and restored the expression of the M572L mutant when overexpressed. The restoration of M572L mutant by Cdc37 required the function of HSP90. These results indicate that Cdc37 in conjunction with HSP90 functions as a molecular chaperone for a temperature-sensitive kinase domain mutant of ZAP70.     

The Schizosaccharomyces pombe Cdc7 protein kinase required for septum formation is a client protein of Cdc37

Cdc37 is an essential molecular chaperone found in fungi and metazoa whose main specificity is for certain protein kinases. Cdc37 can act as an Hsp90 cochaperone or alone; in yeasts, the interaction with Hsp90 is weak and appears not to be essential for Cdc37 function. Numerous genetic interactions between Cdc37 and likely client proteins have been observed in yeasts, but biochemical confirmation has been reported in only a few cases. We and others have generated and characterized temperature-sensitive cdc37 alleles in S. pombe and have used them to investigate the cellular roles of Cdc37: previous work has shown that mitotic Cdc2 is a major client. In this paper, we describe a screen for mutations synthetically lethal with a cdc37ts mutant with the aim of identifying genes encoding further client proteins of Cdc37. Ten such strains were isolated, and genomic libraries were screened for rescuing plasmids. In one case, a truncated cdc7 gene was identified. Further experiments showed that the mutation in this strain was indeed in cdc7. Cdc7 is a protein kinase required for septum initiation, and we show that its kinase activity is greatly reduced when Cdc37 function is impaired. Cdc7 normally locates to the spindle pole body during mitosis, and this appears to be unaffected in the cdc37ts mutant. Other evidence suggests that, in addition to mitosis and septum initiation, Cdc37 may also be required for septum cleavage.     

Hsp90·Cdc37 Complexes with Protein Kinases Form Cooperatively with Multiple Distinct Interaction Sites

Protein kinases are the most prominent group of heat shock protein 90 (Hsp90) clients and are recruited to the molecular chaperone by the kinase-specific cochaperone cell division cycle 37 (Cdc37). The interaction between Hsp90 and nematode Cdc37 is mediated by binding of the Hsp90 middle domain to an N-terminal region of Caenorhabditis elegans Cdc37 (CeCdc37). Here we map the binding site by NMR spectroscopy and define amino acids relevant for the interaction between CeCdc37 and the middle domain of Hsp90. Apart from these distinct Cdc37/Hsp90 interfaces, binding of the B-Raf protein kinase to the cochaperone is conserved between mammals and nematodes. In both cases, the C-terminal part of Cdc37 is relevant for kinase binding, whereas the N-terminal domain displaces the nucleotide from the kinase. This interaction leads to a cooperative formation of the ternary complex of Cdc37 and kinase with Hsp90. For the mitogen-activated protein kinase extracellular signal-regulated kinase 2 (Erk2), we observe that certain features of the interaction with Cdc37·Hsp90 are conserved, but the contribution of Cdc37 domains varies slightly, implying that different kinases may utilize distinct variations of this binding mode to interact with the Hsp90 chaperone machinery.     

Structure of an Hsp90-Cdc37-Cdk4 complex

Activation of many protein kinases depends on their interaction with the Hsp90 molecular chaperone system. Recruitment of protein kinase clients to the Hsp90 chaperone system is mediated by the cochaperone adaptor protein Cdc37, which acts as a scaffold, simultaneously binding protein kinases and Hsp90. We have now expressed and purified an Hsp90-Cdc37-Cdk4 complex, defined its stoichiometry, and determined its 3D structure by single-particle electron microscopy. Comparison with the crystal structure of Hsp90 allows us to identify the locations of Cdc37 and Cdk4 in the complex and suggests a mechanism by which conformational changes in the kinase are coupled to the Hsp90 ATPase cycle.     

Cdc37 engages in stable,S14A mutation-reinforced association with the most atypical member of the yeast kinome,Cdk-activating kinase (Cak1)

In most eukaryotes, Cdc37 is an essential chaperone, transiently associating with newly synthesised protein kinases in order to promote their stabilisation and activation. To determine whether the yeast Cdc37 participates in any stable protein interactions in vivo, genomic two-hybrid screens were conducted using baits that are functional as they preserve the integrity of the conserved N-terminal region of Cdc37, namely a Cdc37-Gal4 DNA binding domain (BD) fusion in both its wild type and its S14 nonphosphorylatable (Cdc37(S14A)) mutant forms. While this failed to identify the protein kinases previously identified as Cdc37 interactors in pull-down experiments, it did reveal Cdc37 engaging in a stable association with the most atypical member of the yeast kinome, cyclin-dependent kinase (Cdk1)-activating kinase (Cak1). Phosphorylation of the conserved S14 of Cdc37 is normally crucial for the interaction with, and stabilisation of, those protein kinase targets of Cdc37, Cak1 is unusual in that the lack of this Cdc37 S14 phosphorylation both reinforces Cak1:Cdc37 interaction and does not compromise Cak1 expression in vivo. Thus, this is the first Cdc37 client kinase found to be excluded from S14 phosphorylation-dependent interaction. The unusual stability of this Cak1:Cdc37 association may partly reflect unique structural features of the fungal Cak1.     

CK2 controls multiple protein kinases by phosphorylating a kinase-targeting molecular chaperone, Cdc37

Cdc37 is a kinase-associated molecular chaperone whose function in concert with Hsp90 is essential for many signaling protein kinases. Here, we report that mammalian Cdc37 is a pivotal substrate of CK2 (casein kinase II). Purified Cdc37 was phosphorylated in vitro on a conserved serine residue, Ser13, by CK2. Moreover, Ser13 was the unique phosphorylation site of Cdc37 in vivo. Crucially, the CK2 phosphorylation of Cdc37 on Ser13 was essential for the optimal binding activity of Cdc37 toward various kinases examined, including Raf1, Akt, Aurora-B, Cdk4, Src, MOK, MAK, and MRK. In addition, nonphosphorylatable mutants of Cdc37 significantly suppressed the association of Hsp90 with protein kinases, while the Hsp90-binding activity of the mutants was unchanged. The treatment of cells with a specific CK2 inhibitor suppressed the phosphorylation of Cdc37 in vivo and reduced the levels of Cdc37 target kinases. These results unveil a regulatory mechanism of Cdc37, identify a novel molecular link between CK2 and many crucial protein kinases via Cdc37, and reveal the molecular basis for the ability of CK2 to regulate pleiotropic cellular functions.     

SLK1, a yeast homolog of MAP kinase activators, has a RAS/cAMP-independent role in nutrient sensing

The Saccharomyces cerevisiae SLK1 protein is implicated in nutrient sensing and growth control. Under nutrient-limiting conditions, slk1 mutants fail to undergo cell cycle arrest. The role of the SLK1 protein in nutrient sensing was examined with respect to the cAMP-dependent protein kinase (PKA) pathway, which has a well characterized role in growth control in yeast, and by the analysis of dominant SLK1 alleles that affect the nutrient response of wild-type cells. Interactions with the PKA pathway were examined by phenotypic analysis of double mutants of slk1 and various PKA pathway mutants. Combining the slk1- mutation with a mutation that is thought constitutively activate the PKA pathway, pde2, resulted in enhanced growth control defects. The combination of slk1- with mutations that inhibit the PKA pathway, cdc25 and ras1 ras2, failed to alleviate the slk1 cell cycle arrest defect and lowered the permissive temperature for growth. Furthermore bcy1 tpk1 tpk2 tpk3 ^w (bcyl tpk ^w) mutants, which have constitutive, low-level, cAMP-independent kinase activity, exhibit nutrient sensing, which is eliminated in the slk1 bcy1 tpk ^w mutants. These results implicated SLK1 in PKA-independent growth control in yeast. The amino-terminal, noncatalytic region of the SLK1 protein may be important in the regulation of SLK1 function in growth control. Overexpression of this region caused starvation sensitivity in wild-type cells by interfering with SLK1 protein function.     

Cdc37 goes beyond Hsp90 and kinases

Cdc37 is a relatively poorly conserved and yet essential molecular chaperone. It has long been thought to function primarily as an accessory factor for Hsp90, notably directing Hsp90 to kinases as substrates. More recent discoveries challenge this simplistic view. Cdc37 client proteins other than kinases have now been found, and Cdc37 displays a variety of Hsp90-independent activities both in vitro and in vivo. It can function as a molecular chaperone by itself, interact with other Hsp90 cochaperones in the absence of Hsp90, and even support yeast growth and protein folding without its Hsp90-binding domain. Thus, for many substrates, there may be many alternative chaperone pathways involving Cdc37, Hsp90, or both.     

Role of Sch9 in regulating Ras-cAMP signal pathway in Saccharomyces cerevisiae

In Saccharomyces cerevisiae PKA plays a major role in regulating cell growth, metabolism, and stress resistance. We report that Sch9 regulates PKA directly and SCH9 deletion enhances PKA activity by showing that: (1) Bcy1 predominately localized in the nucleus in glycerol-grown sch9Δ cells; (2) large part of the catalytic subunits of PKA transferred from the nucleus to the cytoplasm in sch9Δ cells; (3) higher protein stability of Tpk2 resulted in higher protein level of Tpk2 in sch9Δ than in wild type cells. Our investigations suggest that Sch9 regulates phosphorylation of Bcy1. We also observed hyper-phosphorylation of Cdc25 in sch9Δ, in contrast to the tpk2Δ and tpk2Δsch9Δ mutants, suggesting that feedback inhibition of PKA on Cdc25 is through Tpk2.     

Hsp90 and Cdc37 -- a chaperone cancer conspiracy

The Hsp90 molecular chaperone system is involved in the activation of an important set of cell regulatory proteins, including many whose disregulation drives cancer. Recruitment of protein kinases to the Hsp90 system is mediated by the co-chaperone adaptor Cdc37 -- an essential protein whose overexpression is itself, oncogenic. Current structural, biochemical and biological studies of Cdc37 are beginning to unravel the nature of its interactions with Hsp90 and protein kinase clients, and implicate it as a key permissive factor in cell transformation by disregulated protein kinases. The central role of the Hsp90-Cdc37 chaperone complex makes it an important target for future anti-cancer drug development.     

辅助伴侣分子Cdc37蛋白的研究进展

细胞分裂周期蛋白Cdc37最初是在芽殖酵母中发现的细胞周期相关蛋白。随后的研究表明Cdc37具有伴侣分子活性,可以特异地募集一系列的蛋白激酶结合到热激蛋白90（Hsp90）上,形成特定的分子伴侣复合体,参与维持蛋白的稳定性和激酶活性。Cdc37参与了细胞内的多项生命活动,在细胞周期、信号转导和基因表达中都起着重要的作用。由于Cdc37在肿瘤组织中特异性地高表达,使其成为肿瘤治疗中一个重要的分子靶点。     

Cdc37 has distinct roles in protein kinase quality control that protect nascent chains from degradation and promote posttranslational maturation

Cdc37 is a molecular chaperone that functions with Hsp90 to promote protein kinase folding. Analysis of 65 Saccharomyces cerevisiae protein kinases ( approximately 50% of the kinome) in a cdc37 mutant strain showed that 51 had decreased abundance compared with levels in the wild-type strain. Several lipid kinases also accumulated in reduced amounts in the cdc37 mutant strain. Results from our pulse-labeling studies showed that Cdc37 protects nascent kinase chains from rapid degradation shortly after synthesis. This degradation phenotype was suppressed when cdc37 mutant cells were grown at reduced temperatures, although this did not lead to a full restoration of kinase activity. We propose that Cdc37 functions at distinct steps in kinase biogenesis that involves protecting nascent chains from rapid degradation followed by its folding function in association with Hsp90. Our studies demonstrate that Cdc37 has a general role in kinome biogenesis.     

Blocking the chaperone kinome pathway: mechanistic insights into a novel dual inhibition approach for supra-additive suppression of malignant tumors

The chaperone Hsp90 is involved in regulating the stability and activation state of more than 200 ‘client’ proteins and takes part in the cancer diseased states. The major clientele-protein kinases depend on Hsp90 for their proper folding and functioning. Cdc37, a kinase targeting co-chaperone of Hsp90, mediates the interactions between Hsp90 and protein kinases. Targeting of Cdc37 has the prospect of delivering predominantly kinase-selective molecular responses as compared to the current pharmacologic Hsp90 inhibitors. The present work reports a bio-computational study carried out with the aim of exploring the dual inhibition of Hsp90/Cdc37 chaperone/co-chaperone association complex by the naturally occurring drug candidates withaferin A and 17-DMAG along with their possible modes of action. Our molecular docking studies reveal that withaferin A in combination with 17-DMAG can act as potent chaperone system inhibitors. The structural and thermodynamic stability of the ligands’ bound complex was also observed from molecular dynamics simulations in water. Our results suggest a novel tumor suppressive action mechanism of herbal ligands which can be looked forward for further clinical investigations for possible anticancer drug formulations.     

Suppression of a yeast cyclic AMP-dependent protein kinase defect by overexpression of SOK1, a yeast gene exhibiting sequence similarity to a developmentally regulated mouse gene.

Saccharomyces cerevisiae cyclic AMP-dependent protein kinase (A kinase) activity is essential for growth and cell cycle progression. Dependence on A kinase function can be partially relieved by the inactivation of a second kinase encoded by the gene YAK1. We have isolated two new genes, SOK1 and SOK2 (suppressor of kinase), as gene dosage suppressors of the conditional growth defect of several temperature-sensitive A kinase mutants. Overexpression of SOK1, like lesions in YAK1, also restores growth to a strain (tpk1 tpk2 tpk3) lacking all A kinase activity. The SOK1 gene is not essential, but a sok1::HIS3 disruption abrogates suppression of an A kinase defect by yak1. These results suggest that Yak1 and Sok1 define a linear pathway that is partially redundant with that of the A kinase. Activation of Sok1, by SOK1 overexpression or by inactivation of the negative regulator Yak1, renders a cell independent of A kinase function. The implications of such a model are particularly intriguing in light of the nuclear localization pattern of the overexpressed Sok1 protein and the primary sequence homology between SOK1 and a recently described, developmentally regulated mouse gene.     

Structural characterization of the N-terminal kinase-interacting domain of an Hsp90-cochaperone Cdc37 by CD and solution NMR spectroscopy

Cdc37 is a protein kinase-targeting molecular chaperone, which cooperates with Hsp90 to assist the folding, assembly and maturation of various signaling kinases. It consists of three distinct domains: the N-terminal, middle, and C-terminal domain. While the middle domain is an Hsp90-binding domain, the N-terminal domain is recognized as a kinase-interacting domain. The N-terminal domain contains a well-conserved Ser residue at position 13, and the phosphorylation at this site has been shown to be a prerequisite for the interaction between Cdc37 and signaling kinases. Although the phosphorylation of Ser13 might induce some conformational change in Cdc37 molecule, little is known about the structure of the N-terminal domain of Cdc37. We examined the structural and dynamic properties of several fragment proteins corresponding to the N-terminal region of Cdc37 by circular dichroism and solution NMR spectroscopy. We found that the N-terminal domain of Cdc37 exhibits highly dynamic structure, and it exists in the equilibrium between α-helical and more disordered structures. We also found that phosphorylation at Ser13 did not significantly change the overall structure of N-terminal fragment protein of Cdc37. The results suggested that more complicated mechanisms might be necessary to explain the phosphorylation-activated interaction of Cdc37 with various kinases.     

Identification of Cdc37 as a novel regulator of the stress-responsive mitogen-activated protein kinase

Eukaryotic cells utilize multiple mitogen-activated protein kinases (MAPKs) to transmit various extracellular stimuli to the nucleus. A subfamily of MAPKs that mediates environmental stress stimuli is also called stress-activated protein kinase (SAPK), which has crucial roles in cellular survival under stress conditions as well as inflammatory responses. Here we report that Cdc37, an evolutionarily conserved kinase-specific chaperone, is a positive regulator of Spc1 SAPK in the fission yeast Schizosaccharomyces pombe. Through a genetic screen, we have identified cdc37 as a mutation that compromises signaling through Spc1 SAPK. The Cdc37 protein physically interacts with Spc1, and the cdc37 mutation affects both the cellular level of the Spc1 protein and stress-induced Spc1 phosphorylation by Wis1 MAPK kinase (MAPKK). Consistently, expression of the stress response genes regulated by the Spc1 pathway is compromised in cdc37 mutant cells. On the other hand, a mutation in Hsp90, which often cooperates with Cdc37 in chaperoning protein kinases, does not affect Spc1 SAPK. These results suggest that Spc1 SAPK is a novel client protein for the Cdc37 chaperone, and the Cdc37 function is important to maintain the stability of the Spc1 protein and to facilitate stress signaling from Wis1 MAPKK to Spc1 SAPK.     

Cell Surface Cdc37 Participates in Extracellular HSP90 Mediated Cancer Cell Invasion

Cdc37 is a 50 kDa molecular chaperone which targets intrinsically unstable protein kinases to the molecular chaperone HSP90. It is also an over-expressed oncoprotein that mediates carcinogenesis and maintenance of the malignant phenotype by stabilizing the compromised structures of mutant and/or over-expressed oncogenic kinases. Here we report that Cdc37 is not restricted intracellularly but instead it is also present on the surface of MDA-MB-453 and MDA-MB-231 human breast cancer cells, where it is shown to participate in cancer cell motility processes. Furthermore, we demonstrate using an anti-Cdc37 cell impermeable antibody, that similarly to its intracellular counterpart, this surface pool of Cdc37 specifically interacts with HSP90 as well as the kinase receptors HER2 and EGFR on the cell surface, probably acting as a co-factor in HSP90's extracellular chaperoning activities. Finally, we show that functional inhibition of surface HSP90 using mAb 4C5, a cell impermeable monoclonal antibody against this protein, leads not only to disruption of the Cdc37/HSP90 complex but also to inhibition of the Cdc37/ErbB receptors complexes. These results support an essential role for surface Cdc37 in concert with HSP90 on the cell surface during cancer cell invasion processes and strengthen the therapeutic potential of mAb 4C5 for the treatment of cancer.     

Dynamic Tyrosine Phosphorylation Modulates Cycling of the HSP90-P50(CDC37)-AHA1 Chaperone Machine

Many critical protein kinases rely on the Hsp90 chaperone machinery for stability and function. After initially forming a ternary complex with kinase client and the cochaperone p50(Cdc37), Hsp90 proceeds through a cycle of conformational changes facilitated by ATP binding and hydrolysis. Progression through the chaperone cycle requires release of p50(Cdc37) and recruitment of the ATPase activating cochaperone AHA1, but the molecular regulation of this complex process at the cellular level is poorly understood. We demonstrate that a series of tyrosine phosphorylation events, involving both p50(Cdc37) and Hsp90, are minimally sufficient to provide directionality to the chaperone cycle. p50(Cdc37) phosphorylation on Y4 and Y298 disrupts client-p50(Cdc37) association, while Hsp90 phosphorylation on Y197 dissociates p50(Cdc37) from Hsp90. Hsp90 phosphorylation on Y313 promotes recruitment of AHA1, which stimulates Hsp90 ATPase activity, furthering the chaperoning process. Finally, at completion of the chaperone cycle, Hsp90 Y627 phosphorylation induces dissociation of the client and remaining cochaperones.     

Ydj1 protects nascent protein kinases from degradation and controls the rate of their maturation

Ydj1 is a Saccharomyces cerevisiae Hsp40 molecular chaperone that functions with Hsp70 to promote polypeptide folding. We identified Ydj1 as being important for maintaining steady-state levels of protein kinases after screening several chaperones and cochaperones in gene deletion mutant strains. Pulse-chase analyses revealed that a portion of Tpk2 kinase was degraded shortly after synthesis in a ydj1Delta mutant, while the remainder was capable of maturing but with reduced kinetics compared to the wild type. Cdc28 maturation was also delayed in the ydj1Delta mutant strain. Ydj1 protects nascent kinases in different contexts, such as when Hsp90 is inhibited with geldanamycin or when CDC37 is mutated. The protective function of Ydj1 is due partly to its intrinsic chaperone function, but this is minor compared to the protective effect resulting from its interaction with Hsp70. SIS1, a type II Hsp40, was unable to suppress defects in kinase accumulation in the ydj1Delta mutant, suggesting some specificity in Ydj1 chaperone action. However, analysis of chimeric proteins that contained the chaperone modules of Ydj1 or Sis1 indicated that Ydj1 promotes kinase accumulation independently of its client-binding specificity. Our results suggest that Ydj1 can both protect nascent chains against degradation and control the rate of kinase maturation.     

Cdc37 interacts with the glycine-rich loop of Hsp90 client kinases

Recently, we identified a client-binding site of Cdc37 that is required for its association with protein kinases. Phage display technology and liquid chromatography-tandem mass spectrometry (which identifies a total of 33 proteins) consistently identify a unique sequence, GXFG, as a Cdc37-interacting motif that occurs in the canonical glycine-rich loop (GXGXXG) of protein kinases, regardless of their dependence on Hsp90 or Cdc37. The glycine-rich motif of Raf-1 (GSGSFG) is necessary for its association with Cdc37; nevertheless, the N lobe of Raf-1 (which includes the GSGSFG motif) on its own cannot interact with Cdc37. Chimeric mutants of Cdk2 and Cdk4, which differ sharply in their affinities toward Cdc37, show that their C-terminal portions may determine this difference. In addition, a nonclient kinase, the catalytic subunit of cyclic AMP-dependent protein kinase, interacts with Cdc37 but only when a threonine residue in the activation segment of its C lobe is unphosphorylated. Thus, although a region in the C termini of protein kinases may be crucial for accomplishing and maintaining their interaction with Cdc37, we conclude that the N-terminal glycine-rich loop of protein kinases is essential for physically associating with Cdc37.