Invariant Leu preceding turn motif phosphorylation site controls the interaction of protein kinase C with Hsp70

Heat shock proteins play important roles in regulating signal transduction in cells by associating with, and stabilizing, diverse signaling molecules, including protein kinases. Previously, we have shown that heat shock protein Hsp70 associates with protein kinase C (PKC) via an interaction that is triggered by dephosphorylation at the turn phosphorylation motif. Here we have identified an invariant residue in the carboxyl terminus of PKC that mediates the binding to Hsp70. Specifically, we show that Hsp70 binds to Leu (Leu-640) immediately preceding the conserved turn motif autophosphorylation site (Thr-641) in PKC betaII. Co-immunoprecipitation experiments reveal that mutation of Leu-640 to Gly decreases the interaction of Hsp70 with PKC betaII. This weakened interaction between Hsp70 and the mutant PKCs results in accumulation of dephosphorylated PKC in the detergent-insoluble fraction of cells. In addition, the Hsp70-binding mutant is considerably more sensitive to down-regulation compared with WT PKC: disruption of Hsp70 binding leads to accelerated dephosphorylation and enhanced ubiquitination of mutant PKC upon phorbol ester treatment. Last, pulse-chase experiments demonstrate that Hsp70 preferentially binds the species of mature PKC that has become dephosphorylated compared with the newly synthesized protein that has yet to be phosphorylated. Thus, Hsp70 binds a hydrophobic residue preceding the turn motif, protecting PKC from down-regulation and sustaining the signaling lifetime of the kinase.     

Characterization of Celastrol to Inhibit Hsp90 and Cdc37 Interaction

The molecular chaperone heat shock protein 90 (Hsp90) is required for the stabilization and conformational maturation of various oncogenic proteins in cancer. The loading of protein kinases to Hsp90 is actively mediated by the cochaperone Cdc37. The crucial role of the Hsp90-Cdc37 complex has made it an exciting target for cancer treatment. In this study, we characterize Hsp90 and Cdc37 interaction and drug disruption using a reconstituted protein system. The GST pull-down assay and ELISA assay show that Cdc37 binds to ADP-bound/nucleotide-free Hsp90 but not ATP-bound Hsp90. Celastrol disrupts Hsp90-Cdc37 complex formation, whereas the classical Hsp90 inhibitors (e.g. geldanamycin) have no effect. Celastrol inhibits Hsp90 ATPase activity without blocking ATP binding. Proteolytic fingerprinting indicates celastrol binds to Hsp90 C-terminal domain to protect it from trypsin digestion. These data suggest that celastrol may represent a new class of Hsp90 inhibitor by modifying Hsp90 C terminus to allosterically regulate its chaperone activity and disrupt Hsp90-Cdc37 complex.     

The carboxyl terminus of protein kinase c provides a switch to regulate its interaction with the phosphoinositide-dependent kinase, PDK-1

The function of protein kinase C family members depends on two tightly coupled phosphorylation mechanisms: phosphorylation of the activation loop by the phosphoinositide-dependent kinase, PDK-1, followed by autophosphorylation at two positions in the COOH terminus, the turn motif, and the hydrophobic motif. Here we address the molecular mechanisms underlying the regulation of protein kinase C betaII by PDK-1. Co-immunoprecipitation studies reveal that PDK-1 associates preferentially with its substrate, unphosphorylated protein kinase C, by a direct mechanism. The exposed COOH terminus of protein kinase C provides the primary interaction site for PDK-1, with co-expression of constructs of the carboxyl terminus effectively disrupting the interaction in vivo. Disruption of this interaction promotes the autophosphorylation of protein kinase C, suggesting that the binding of PDK-1 to the carboxyl terminus protects it from autophosphorylation. Studies with constructs of the COOH terminus reveal that the intrinsic affinity of PDK-1 for phosphorylated COOH terminus is over an order of magnitude greater than that for unphosphorylated COOH terminus, contrasting with the finding that PDK-1 does not bind phosphorylated protein kinase C effectively. However, effective binding of the phosphorylated species can be induced by the activated conformation of protein kinase C. This suggests that the carboxyl terminus becomes masked following autophosphorylation, a process that can be reversed by the conformational changes accompanying activation. Our data suggest a model in which PDK-1 provides two points of regulation of protein kinase C: 1) phosphorylation of the activation loop, which is regulated by the intrinsic activity of PDK-1, and 2) phosphorylation of the carboxyl terminus, which is regulated by the release of PDK-1 to allow autophosphorylation.     

The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins

To maintain quality control in cells, mechanisms distinguish among improperly folded peptides, mature and functional proteins, and proteins to be targeted for degradation. The molecular chaperones, including heat-shock protein Hsp90, have the ability to recognize misfolded proteins and assist in their conversion to a functional conformation. Disruption of Hsp90 heterocomplexes by the Hsp90 inhibitor geldanamycin leads to substrate degradation through the ubiquitin-proteasome pathway, implicating this system in protein triage decisions. We previously identified CHIP (carboxyl terminus of Hsc70-interacting protein) to be an interaction partner of Hsc70 (ref. 4). CHIP also interacts directly with a tetratricopeptide repeat acceptor site of Hsp90, incorporating into Hsp90 heterocomplexes and eliciting release of the regulatory cofactor p23. Here we show that CHIP abolishes the steroid-binding activity and transactivation potential of the glucocorticoid receptor, a well-characterized Hsp90 substrate, even though it has little effect on its synthesis. Instead, CHIP induces ubiquitylation of the glucocorticoid receptor and degradation through the proteasome. By remodelling Hsp90 heterocomplexes to favour substrate degradation, CHIP modulates protein triage decisions that regulate the balance between protein folding and degradation for chaperone substrates.     

Hsp90 functions to balance the phosphorylation state of Akt during C2C12 myoblast differentiation

The function of the 90-kDa heat shock protein (Hsp90) is essential for the regulation of a myriad of signal transduction cascades that control all facets of a cell's physiology. Akt (PKB) is an Hsp90-dependent serine-threonine kinase that plays critical roles in the regulation of muscle cell physiology, including roles in the regulation of muscle differentiation and anti-apoptotic responses that modulate cell survival. In this report, we have examined the role of Hsp90 in regulating the activity of Akt in differentiating C2C12 myoblasts. While long-term treatment of differentiating C2C12 cells with the Hsp90 inhibitor geldanamycin led to the depletion of cellular Akt levels, pulse-chase analysis indicated that geldanamycin primarily enhanced the turnover rate of newly synthesized Akt. Hsp90 maintained an interaction with mature Akt, while Cdc37, Hsp90's kinase-specific co-chaperone, was lost from the chaperone complex upon Akt maturation. Geldanamycin partially disrupted the interaction of Cdc37 with Akt, but had a much less significant effect on the interaction of Hsp90 with Akt. Surprisingly, short-term treatment of differentiating C2C12 with geldanamycin increased the phosphorylation of Akt on Ser473, an effect mimicked by treatment of C2C12 cells with okadaic acid or the Hsp90 inhibitor novobiocin. Furthermore, Akt was found to interact directly with catalytic subunit of protein phosphatase 2A (PP2Ac) in C2C12 cells, and this interaction was not disrupted by geldanamycin. Thus, our findings indicate that Hsp90 functions to balance the phosphorylation state of Akt by modulating the ability of Akt to be dephosphorylated by PP2Ac during C2C12 myoblast differentiation.     

CAIR-1/BAG-3 abrogates heat shock protein-70 chaperone complex-mediated protein degradation: accumulation of poly-ubiquitinated Hsp90 client proteins

BAG family proteins are regulatory co-chaperones for heat shock protein (Hsp) 70. Hsp70 facilitates the removal of injured proteins by ubiquitin-mediated proteasomal degradation. This process can be driven by geldanamycin, an irreversible blocker of Hsp90. We hypothesize that CAIR-1/BAG-3 inhibits Hsp-mediated proteasomal degradation. Human breast cancer cells were engineered to overexpress either full-length CAIR-1 (FL), which binds Hsp70, or a BAG domain-deletion mutant (dBAG) that cannot bind Hsp70. FL overexpression prevented geldanamycin-mediated loss of total and phospho-Akt and other Hsp client proteins. dBAG provided no protection, indicating a requirement for Hsp70 binding. Ubiquitinated Akt accumulated in FL-expressing cells, mimicking the effect of lactacystin proteasomal inhibition, indicating that CAIR-1 inhibits proteasomal degradation distal to protein ubiquitination in a BAG domain-dependent manner. Protein protection in FL cells was generalizable to downstream Akt targets, GSK3beta, P70S6 kinase, CREB, and other Hsp client proteins, including Raf-1, cyclin-dependent kinase 4, and epidermal growth factor receptor. These findings suggest that Hsp70 is a chaperone driving a multiprotein degradation complex and that the inhibitory co-chaperone CAIR-1 functions distal to client ubiquitination. Furthermore, poly-ubiquitination is not sufficient for efficient proteasomal targeting of Hsp client proteins.     

Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein

To elucidate the function of keratins 8 and 18 (K8/18), major components of the intermediate filaments of simple epithelia, we searched for K8/18-binding proteins by screening a yeast two-hybrid library. We report here that human Mrj, a DnaJ/Hsp40 family protein, directly binds to K18. Among the interactions between DnaJ/Hsp40 family proteins and various intermediate filament proteins that we tested using two-hybrid methods, Mrj specifically interacted with K18. Immunostaining with anti-Mrj antibody showed that Mrj colocalized with K8/18 filaments in HeLa cells. Mrj was immunoprecipitated not only with K18, but also with the stress-induced and constitutively expressed heat shock protein Hsp/c70. Mrj bound to K18 through its C terminus and interacted with Hsp/c70 via its N terminus, which contains the J domain. Microinjection of anti-Mrj antibody resulted in the disorganization of K8/18 filaments, without effects on the organization of actin filaments and microtubules. Taken together, these results suggest that Mrj may play an important role in the regulation of K8/18 filament organization as a K18-specific co-chaperone working together with Hsp/c70.     

A proteomic screen identified stress-induced chaperone proteins as targets of Akt phosphorylation in mesangial cells

The serine-threonine kinase Akt regulates mesangial cell apoptosis, proliferation, and hypertrophy. To define Akt signaling pathways in mesangial cells, we performed a functional proteomic screen for rat mesangial cell proteins phosphorylated by Akt. A group of chaperone proteins, heat shock protein (Hsp) 70, Hsp90alpha, Hsp90beta, Glucose-regulated protein (Grp) Grp78, Grp94, and protein disulfide isomerase (PDI) were identified as potential Akt substrates by two techniques: (a) in vitro phosphorylation of mesangial cell lysate by recombinant active Akt followed by protein separation by SDS-PAGE or 2-DE and phosphoprotein identification by peptide mass fingerprinting using MALDI-MS, or (b) immunoblot analysis of proteins from PDGF-stimulated mesangial cells using an anti-Akt phospho-motif antibody. In vitro kinase reactions using recombinant proteins confirmed that Akt phosphorylates Hsp70, Hsp90alpha and beta, Grp94, and PDI. Immunoprecipitation of Akt from mesangial cell lysate coprecipitated Grp78 and Hsp70. PDGF stimulation of mesangial cells caused an acidic shift in the isoelectric point of Hsp70, Hsp90, and PDI that was dependent on PI-3K activity for Hsp70 and Hsp90. The data suggest that Akt-mediated phosphorylation of stress-induced chaperones represents a mechanism for regulation of chaperone function during mesangial cell responses to physiologic and pathologic stimuli.     

Identification of CHIP, a Novel Tetratricopeptide Repeat-Containing Protein That Interacts with Heat Shock Proteins and Negatively Regulates Chaperone Functions

The chaperone function of the mammalian 70-kDa heat shock proteins Hsc70 and Hsp70 is modulated by physical interactions with four previously identified chaperone cofactors: Hsp40, BAG-1, the Hsc70-interacting protein Hip, and the Hsc70-Hsp90-organizing protein Hop. Hip and Hop interact with Hsc70 via a tetratricopeptide repeat domain. In a search for additional tetratricopeptide repeat-containing proteins, we have identified a novel 35-kDa cytoplasmic protein, carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP is highly expressed in adult striated muscle in vivo and is expressed broadly in vitro in tissue culture. Hsc70 and Hsp70 were identified as potential interaction partners for this protein in a yeast two-hybrid screen. In vitro binding assays demonstrated direct interactions between CHIP and both Hsc70 and Hsp70, and complexes containing CHIP and Hsc70 were identified in immunoprecipitates of human skeletal muscle cells in vivo. Using glutathione S-transferase fusions, we found that CHIP interacted with the carboxy-terminal residues 540 to 650 of Hsc70, whereas Hsc70 interacted with the amino-terminal residues 1 to 197 (containing the tetratricopeptide domain and an adjacent charged domain) of CHIP. Recombinant CHIP inhibited Hsp40-stimulated ATPase activity of Hsc70 and Hsp70, suggesting that CHIP blocks the forward reaction of the Hsc70-Hsp70 substrate-binding cycle. Consistent with this observation, both luciferase refolding and substrate binding in the presence of Hsp40 and Hsp70 were inhibited by CHIP. Taken together, these results indicate that CHIP decreases net ATPase activity and reduces chaperone efficiency, and they implicate CHIP in the negative regulation of the forward reaction of the Hsc70-Hsp70 substrate-binding cycle.     

Heat shock protein 27 association with the I kappa B kinase complex regulates tumor necrosis factor alpha-induced NF-kappa B activation

Heat shock protein 27 (Hsp27) is a ubiquitously expressed member of the heat shock protein family that has been implicated in various biological functions including the response to heat shock, oxidative stress, and cytokine treatment. Previous studies have demonstrated that heat shock proteins are involved in regulating signal transduction pathways including the NF-kappa B pathway. In this study, we demonstrated that Hsp27 associates with the I kappa B kinase (IKK) complex and that this interaction was stimulated by tumor necrosis factor alpha treatment. Phosphorylation of Hsp27 by the kinase mitogen-activated protein kinase-activated protein kinase 2, a downstream substrate of the mitogen-activated protein kinase p38, enhanced the association of Hsp27 with IKK beta to result in decreased IKK activity. Consistent with these observations, treatment of cells with a p38 inhibitor reduced the association of Hsp27 with IKK beta and thus resulted in increased IKK activity. These studies indicate that Hsp27 plays a negative role in down-regulating IKK signaling by reducing its activity following tumor necrosis factor alpha stimulation.     

Transforming growth factor-beta 1 rapidly induces Hsp70 and Hsp90 molecular chaperones in cultured chicken embryo cells.

In this report we show that: (1) molecular chaperones in the heat shock protein (hsp) family are a new class of cellular proteins induced by Transforming Growth Factor-beta 1 (TGF beta), a cytokine present in serum, (2) rapid induction of Hsc70 precedes a general increase in protein synthesis and may be a preparatory event, (3) TGF beta is a potent regulator of overall protein synthesis in chicken embryo cells (CEC), and (4) isoforms of Hsp90 with different biochemical properties exist, raising the possibility that they may have different functions. TGF beta can substitute for serum in stimulating synthesis of members of the Hsp90 and Hsp70 families of stress proteins, whereas other cytokines, including PDGF, FGF, and EGF, were not effective nor did they enhance the stimulatory effect of TGF beta on the hsp's. Analysis of the induction of hsp's using one- and two-dimensional polyacrylamide gel electrophoresis indicated that members of the Hsp70 family of molecular chaperones were induced rapidly by TGF beta, reaching maximum rates of accumulation by 5 hours of treatment. Total protein synthesis increased more slowly, undergoing an approximately twofold increase in 24 hours. Using a modified protocol for two-dimensional gel electrophoresis, the Hsp90 protein family was separated into four isoelectric forms, two of which were phosphorylated (Hsp90-2 and -4). These phosphorylated isoforms turned over faster than the unphosphorylated forms of Hsp90. All four isoforms were heat inducible, but only Hsp90-2 and -3 were induced rapidly by TGF beta, again within 5 hours of treatment. The effects of serum on these protein families were similar to those of TGF beta, suggesting that this cytokine may be the serum component primarily responsible for up-regulating members of the Hsp90 and Hsp70 families. We hypothesize that cells rapidly increase their chaperoning capacity for newly synthesized polypeptides in preparation for an increase in the rate of synthesis of proteins up-regulated by TGF beta.     

Endoplasmic Reticulum-associated Degradation of Niemann-Pick C1: EVIDENCE FOR THE ROLE OF HEAT SHOCK PROTEINS AND IDENTIFICATION OF LYSINE RESIDUES THAT ACCEPT UBIQUITIN*

Most cases with Niemann-Pick disease type C carry mutations in NPC1. Some of the mutations, including the most frequent I1061T, give rise to unstable proteins selected for endoplasmic reticulum-associated degradation. The purpose of the current study was to shed mechanistic insights into the degradation process. A proteasome inhibitor MG132 prolonged the life span of the wild-type NPC1 expressed in COS cells. The expressed protein associated with multiple chaperones including heat shock protein 90 (Hsp90), Hsp70, heat shock cognate protein 70 (Hsc70), and calnexin. Accordingly, expression of an E3 ligase CHIP (carboxyl terminus of Hsp70-interacting protein) enhanced MG132-induced accumulation of ubiquitylated NPC1. Co-expression and RNAi knockdown experiments in HEK cells indicated that Hsp70/Hsp90 stabilized NPC1, whereas Hsc70 destabilized it. In human fibroblasts carrying the I1061T mutation, adenovirus-mediated expression of Hsp70 or treatment with an HSP-inducer geranylgeranylacetone (GGA) increased the level of the mutant protein. In GGA-treated cells, the rescued protein was localized in the late endosome and ameliorated cholesterol accumulation. MALDI-TOF mass spectrometry revealed three lysine residues at amino acids 318, 792, and 1180 as potential ubiquitin-conjugation sites. Substitutions of the three residues with alanine yielded a mutant protein with a steady-state level more than three times higher than that of the wild-type. Introduction of the same substitutions to the I1061T mutant resulted in an increase in its protein level and functional restoration. These findings indicated the role of HSPs in quality control of NPC1 and revealed the role of three lysine residues as ubiquitin-conjugation sites.     

Understanding the Functional Interplay between Mammalian Mitochondrial Hsp70 Chaperone Machine Components

Mitochondria biogenesis requires the import of several precursor proteins that are synthesized in the cytosol. The mitochondrial heat shock protein 70 (mtHsp70) machinery components are highly conserved among eukaryotes, including humans. However, the functional properties of human mtHsp70 machinery components have not been characterized among all eukaryotic families. To study the functional interactions, we have reconstituted the components of the mtHsp70 chaperone machine (Hsp70/J-protein/GrpE/Hep) and systematically analyzed in vitro conditions for biochemical functions. We observed that the sequence-specific interaction of human mtHsp70 toward mitochondrial client proteins differs significantly from its yeast counterpart Ssc1. Interestingly, the helical lid of human mtHsp70 was found dispensable to the binding of P5 peptide as compared with the other Hsp70s. We observed that the two human mitochondrial matrix J-protein splice variants differentially regulate the mtHsp70 chaperone cycle. Strikingly, our results demonstrated that human Hsp70 escort protein (Hep) possesses a unique ability to stimulate the ATPase activity of mtHsp70 as well as to prevent the aggregation of unfolded client proteins similar to J-proteins. We observed that Hep binds with the C terminus of mtHsp70 in a full-length context and this interaction is distinctly different from unfolded client-specific or J-protein binding. In addition, we found that the interaction of Hep at the C terminus of mtHsp70 is regulated by the helical lid region. However, the interaction of Hep at the ATPase domain of the human mtHsp70 is mutually exclusive with J-proteins, thus promoting a similar conformational change that leads to ATPase stimulation. Additionally, we highlight the biochemical defects of the mtHsp70 mutant (G489E) associated with a myelodysplastic syndrome.     

G protein-coupled receptor kinase interaction with Hsp90 mediates kinase maturation

G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine-specific protein kinase that mediates agonist-dependent phosphorylation of numerous G protein-coupled receptors. In an effort to identify proteins that regulate GRK2 function, we searched for interacting proteins by immunoprecipitation of endogenous GRK2 from HL60 cells. Subsequent analysis by gel electrophoresis and mass spectrometry revealed that GRK2 associates with heat shock protein 90 (Hsp90). GRK2 interaction with Hsp90 was confirmed by co-immunoprecipitation and was effectively disrupted by geldanamycin, an Hsp90-specific inhibitor. Interestingly, geldanamycin treatment of HL60 cells decreased the expression of endogenous GRK2 in a dose- and time-dependent manner, and metabolic labeling demonstrated that geldanamycin rapidly accelerated the degradation of newly synthesized GRK2. The use of various protease inhibitors suggested that GRK2 degradation induced by geldanamycin was predominantly through the proteasome pathway. To test whether Hsp90 plays a general role in regulating GRK maturation, additional GRKs were studied by transient expression in COS-1 cells and subsequent treatment with geldanamycin. These studies demonstrate that GRK3, GRK5, and GRK6 are also stabilized by interaction with Hsp90. Taken together, our work revealed that GRK interaction with heat shock proteins plays an important role in regulating GRK maturation.     

beta 1-adrenergic receptor association with the synaptic scaffolding protein membrane-associated guanylate kinase inverted-2 (MAGI-2). Differential regulation of receptor internalization by MAGI-2 and PSD-95

The beta1-adrenergic receptor (beta1AR) is known to be localized to synapses and to modulate synaptic plasticity in many brain regions, but the molecular mechanisms determining beta1AR subcellular localization are not fully understood. Using overlay and pull-down techniques, we found that the beta1AR carboxyl terminus associates with MAGI-2 (membrane-associated guanylate kinase inverted-2), a protein also known as S-SCAM (synaptic scaffolding molecule). MAGI-2 is a multidomain scaffolding protein that contains nine potential protein-protein interaction modules, including 6 PDZ domains, 2 WW domains, and a guanylate kinase-like domain. The beta1AR carboxyl terminus binds with high affinity to the first PDZ domain of MAGI-2, with the last few amino acids of the beta1AR carboxyl terminus being the key determinants of the interaction. In cells, the association of full-length beta1AR with MAGI-2 occurs constitutively and is enhanced by agonist stimulation of the receptor, as assessed by both co-immunoprecipitation experiments and immunofluorescence co-localization studies. Agonist-induced internalization of the beta1AR is markedly increased by co-expression with MAGI-2. Strikingly, this result is the opposite of the effect of co-expression with PSD-95, a previously reported binding partner of the beta1AR. Further cellular experiments revealed that MAGI-2 has no effect on beta1AR oligomerization but does promote association of beta1AR with the cytoplasmic signaling protein beta-catenin, a known MAGI-2 binding partner. These data reveal that MAGI-2 is a specific beta1AR binding partner that modulates beta1AR function and facilitates the physical association of the beta1AR with intracellular proteins involved in signal transduction and synaptic regulation.     

Chaperone-mediated hierarchical control in targeting misfolded proteins to aggresomes

Protein misfolding is a common event in living cells. Molecular chaperones not only assist protein folding; they also facilitate the degradation of misfolded polypeptides. When the intracellular degradative capacity is exceeded, juxtanuclear aggresomes are formed to sequester misfolded proteins. Despite the well-established role of chaperones in both protein folding and degradation, how chaperones regulate the aggregation process remains controversial. Here we investigate the molecular mechanisms underlying aggresome formation in mammalian cells. Analysis of the chaperone requirements for the fate of misfolded proteins reveals an unexpected role of heat shock protein 70 (Hsp70) in promoting aggresome formation. This proaggregation function of Hsp70 relies on the interaction with the cochaperone ubiquitin ligase carboxyl terminal of Hsp70/Hsp90 interacting protein (CHIP). Disrupting Hsp70-CHIP interaction prevents the aggresome formation, whereas a dominant-negative CHIP mutant sensitizes the aggregation of misfolded protein. This accelerated aggresome formation also relies on the stress-induced cochaperone Bcl2-associated athanogene 3. Our results indicate that a hierarchy of cochaperone interaction controls different aspects of the intracellular protein triage decision, extending the function of Hsp70 from folding and degradation to aggregation.     

Role of inducible heat shock protein 70 in radiation-induced cell death

We previously demonstrated the protective effect of inducible heat shock protein 70 (Hsp70) against gamma radiation. Herein, we extend our studies on the possible role of Hsp70 to ionizing radiation-induced cell cycle regulation. The growth rate of inducible hsp70-transfected cells was 2-3 hours slower than that of control cells. Flow cytometric analysis of cells at G1 phase synchronized by serum starvation also showed the growth delay in the Hsp70-overexpressing cells. In addition, reduced cyclin D1 and Cdc2 levels and increased dephosphorylated phosphoretinoblastoma (pRb) were observed in inducible hsp70-transfected cells, which were probably responsible for the reduction of cell growth. To find out if inducible Hsp70-mediated growth delay affected radiation-induced cell cycle regulation, flow cytometric and molecular analyses of cell cycle regulatory proteins and their kinase were performed. The radiation-induced G2/M arrest was found to be inhibited by Hsp70 overexpression and reduced p21Waf induction and its kinase activity by radiation in the Hsp70-transfected cells. In addition, radiation-induced cyclin A or B1 expressions together with their kinase activities were also inhibited by inducible Hsp70, which represented reduced mitotic cell death. Indeed, hsp70 transfectants showed less induction of radiation-induced apoptosis. When treated with nocodazole, radiation-induced mitotic arrest was inhibited by inducible Hsp70. These results strongly suggested that inducible Hsp70 modified growth delay (increased G1 phase) and reduced G2/M phase arrest, subsequently resulting in inhibition of radiation-induced cell death.