Hsp40 facilitates nuclear import of the human immunodeficiency virus type 2 Vpx-mediated preintegration complex

Human immunodeficiency virus type 2 (HIV-2) Vpx is required for nuclear translocation of the viral preintegration complex (PIC) in quiescent cells. In order to decipher the mechanism of action of Vpx, a cDNA library was screened with the yeast two-hybrid assay, resulting in the identification of heat shock protein 40, Hsp40/DnaJB6, as a Vpx-interactive protein. Interaction with Vpx was confirmed by glutathione S-transferase (GST) pull-down and coimmunoprecipitation assays. Overexpression of Hsp40/DnaJB6 enhanced Vpx nuclear import, whereas overexpression of a nuclear localization mutant of Hsp40/DnaJB6 (H31Q) or down-regulation of Hsp40/DnaJB6 by small interfering RNA (siRNA) reduced the nuclear import of Vpx. Hsp40/DnaJB6 competed with the Pr55^Gag precursor protein for the binding of Vpx and incorporation into virus-like particles. Overexpression of Hsp40/DnaJB6 promoted viral PIC nuclear import, whereas siRNA down-regulation of Hsp40/DnaJB6 inhibited PIC nuclear import. These results demonstrate a role for Hsp40/DnaJB6 in the regulation of HIV-2 PIC nuclear transport.     

Modulation of polyglutamine inclusion formation by the Hsp70 chaperone machine

Components of the Hsp70 chaperone machine have been implied in protection against polyglutamine (poly-Q) pathologies. Yet, little is known about specific mechanisms and the rate-limiting components that account for this protective effect. Here, we examined the effects of an Hsp70 chaperone family member (HspA1A) and its cofactors Hsp40 (DnaJB1), Bag-1 and CHIP on poly-Q protein inclusion formation and SDS-insolubilization. Overexpression of HspA1A alone did not suppress inclusion formation, while overexpression of DnaJB1 reduced poly-Q inclusion formation and insolubilization. The reducing effect of DnaJB1 on inclusion formation was enhanced by coexpressing HspA1A, and was dependent on the interaction of DnaJB1 with Hsp70/Hsc70 chaperones. Additionally, two factors connecting Hsp70 activity with protein degradation by the ubiquitin-proteasome system Bag-1 and CHIP slightly decreased the levels of soluble poly-Q protein, but the amount of aggregated protein and fraction of cells with inclusions remained unaltered. Our data suggest that the HspA1A chaperone machine can modulate poly-Q inclusion formation depending on the ratio of its components and that DnaJB1 is the rate-limiting step.     

Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent nuclear import signal.

BACKGROUND: At M phase, cyclin B1 is phosphorylated in the cytoplasmic retention sequence (CRS), which is required for nuclear export. During interphase, cyclin B1 shuttles between the nucleus and the cytoplasm because constitutive nuclear import is counteracted by rapid nuclear export. In M phase, cyclin B moves rapidly into the nucleus coincident with its phosphorylation, an overall movement that might be caused simply by a decrease in its nuclear export. However, the questions of whether CRS phosphorylation is required for cyclin B1 translocation in mitosis and whether a reduction in nuclear export is sufficient to explain its rapid relocalisation have not been addressed. RESULTS: We have used two forms of green fluorescent protein to analyse simultaneously the translocation of wild-type cyclin B1 and a phosphorylation mutant of cyclin B1 in mitosis, and correlated this with an in vitro nuclear import assay. We show that cyclin B1 rapidly translocates into the nucleus approximately 10 minutes before breakdown of the nuclear envelope, and that this movement requires the CRS phosphorylation sites. A cyclin B1 mutant that cannot be phosphorylated enters the nucleus after the wild-type protein. Phosphorylation of the CRS creates a nuclear import signal that enhances cyclin B1 import in vitro and in vivo, in a manner distinct from the previously described import of cyclin B1 mediated by importin beta. CONCLUSIONS: We show that phosphorylation of human cyclin B1 is required for its rapid translocation to the nucleus towards the end of prophase. Phosphorylation enhances cyclin B1 nuclear import by creating a nuclear import signal. The phosphorylation of the CRS is therefore a critical step in the control of mitosis.     

Human Parvovirus B19 Induces Cell Cycle Arrest at G2 Phase with Accumulation of Mitotic Cyclins

Human parvovirus B19 infects specifically erythroid progenitor cells, which causes transient aplastic crises and hemolytic anemias. Here, we demonstrate that erythroblastoid UT7/Epo cells infected with B19 virus fall into growth arrest with 4N DNA, indicating G(2)/M arrest. These B19 virus-infected cells displayed accumulation of cyclin A, cyclin B1, and phosphorylated cdc2 and were accompanied by an up-regulation in the kinase activity of the cdc2-cyclin B1 complex, similar to that in cells treated with the mitotic inhibitor. However, degradation of nuclear lamina and phosphorylation of histone H3 and H1 were not seen in B19 virus-infected cells, indicating that the infected cells do not enter the M phase. Accumulation of cyclin B1 was persistently localized in the cytoplasm, but not in the nucleus, suggesting that B19 virus infection of erythroid cells raises suppression of nuclear import of cyclin B1, resulting in cell cycle arrest at the G(2) phase. The B19 virus-induced G(2)/M arrest may be the critical event in the damage of erythroid progenitor cells seen in patients with B19 virus infection.     

Two distinct classes of cochaperones compete for the EEVD motif in heat shock protein 70 to tune its chaperone activities

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein–protein interactions with many cochaperones. One “hotspot” for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, such as the E3 ubiquitin ligase CHIP. In addition, the EEVD motif also interacts with a structurally distinct domain that is present in class B J-domain proteins, such as DnaJB4. These observations suggest that CHIP and DnaJB4 might compete for binding to Hsp70’s EEVD motif; however, the molecular determinants of such competition are not clear. Using a collection of EEVD-derived peptides, including mutations and truncations, we explored which residues are critical for binding to both CHIP and DnaJB4. These results revealed that some features, such as the C-terminal carboxylate, are important for both interactions. However, CHIP and DnaJB4 also had unique preferences, especially at the isoleucine position immediately adjacent to the EEVD. Finally, we show that competition between these cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70–CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70–DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein–protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded.     

Identification of nuclear-import and cell-cycle regulatory proteins that bind to prothymosin alpha.

Prothymosin alpha (ProT alpha) is a nuclear protein that is widely distributed in mammalian tissues, and is thought to play a role in cell proliferation. In an attempt to shed light on this role, affinity chromatography on ProT alpha-Sepharose columns was used to identify proteins in subcellular extracts of transformed human lymphocytes (NC37 cells) that interact with ProT alpha in vitro, and thus may interact with ProT alpha in vivo. Immunoblotting techniques were used to screen the ProT alpha-binding fractions for histones and other proteins involved in nuclear transport and cell-cycle control. The most abundant ProT alpha-binding proteins were histones H2A, H2B, H3, and H4. Of the nuclear-transport proteins, karyopherin beta1, Rch-1, Ran, and RCC1 were detected at high concentrations; NTF2, nucleoporin p62, and Hsp70 were detected at low concentrations; while tranportin, CAS, and Ran BPI were not detected. Of the cell-cycle control proteins, PCNA, Cdk2, and cyclin A were detected at high concentrations; cdc2, Cdk4, and cyclin B were detected at very low concentrations; while cyclin D1, cyclin D3, Cip1, and Kip1 were not detected. These results suggest (i) that ProT alpha is transported into the nucleus by the karyopherin beta1-Rch-1 complex, and (ii) that ProT alpha may interact in the nucleus with proteins involved in DNA metabolism and cell-cycle control.     

p21-Mediated nuclear retention of cyclin B1-Cdk1 in response to genotoxic stress

G2 arrest of cells suffering DNA damage in S phase is crucial to avoid their entry into mitosis, with the concomitant risks of oncogenic transformation. According to the current model, signals elicited by DNA damage prevent mitosis by inhibiting both activation and nuclear import of cyclin B1-Cdk1, a master mitotic regulator. We now show that normal human fibroblasts use additional mechanisms to block activation of cyclin B1-Cdk1. In these cells, exposure to nonrepairable DNA damage leads to nuclear accumulation of inactive cyclin B1-Cdk1 complexes. This nuclear retention, which strictly depends on association with endogenous p21, prevents activation of cyclin B1-Cdk1 by Cdc25 and Cdk-activating kinase as well as its recruitment to the centrosome. In p21-deficient normal human fibroblasts and immortal cell lines, cyclin B1 fails to accumulate in the nucleus and could be readily detected at the centrosome in response to DNA damage. Therefore, in normal cells, p21 exerts a dual role in mediating DNA damage-induced cell cycle arrest and exit before mitosis. In addition to blocking pRb phosphorylation, p21 directly prevents mitosis by inactivating and maintaining the inactive state of mitotic cyclin-Cdk complexes. This, with subsequent degradation of mitotic cyclins, further contributes to the establishment of a permanent G2 arrest.     

DNA damage-induced nuclear translocation of Apaf-1 is mediated by nucleoporin Nup107

Beside its central role in the mitochondria-dependent cell death pathway, the apoptotic protease activating factor 1 (Apaf-1) is involved in the DNA damage response through cell-cycle arrest induced by genotoxic stress. This non-apoptotic function requires a nuclear translocation of Apaf-1 during the G1-to-S transition. However, the mechanisms that trigger the nuclear accumulation of Apaf-1 upon DNA damage remain to be investigated. Here we show that the main 4 isoforms of Apaf-1 can undergo nuclear translocation and restore Apaf-1 deficient MEFs cell cycle arrest in the S phase following genotoxic stress through activation of Chk-1. Interestingly, DNA damage-dependent nuclear accumulation of Apaf-1 occurs independently of p53 and the retinoblastoma (pRb) pathway. We demonstrated that Apaf-1 associates with the nucleoporin Nup107 and this association is necessary for Apaf-1 nuclear import. The CED-4 domain of Apaf-1 directly binds to the central domain of Nup107 in an ATR-regulated, phosphorylation-dependent manner. Interestingly, expression of the Apaf-1-interacting domain of Nup107 interfered with Apaf-1 nuclear translocation upon genotoxic stress, resulting in a marked reduction of Chk-1 activation and cell cycle arrest. Thus, our results confirm the crucial role of Apaf-1 nuclear relocalization in mediating cell-cycle arrest induced by genotoxic stress and implicate Nup107 as a critical regulator of the DNA damage-induced intra-S phase checkpoint response.     

Hikeshi, a nuclear import carrier for Hsp70s, protects cells from heat shock-induced nuclear damage

During heat shock stress, importin β family-mediated nucleocytoplasmic trafficking is downregulated, whereas nuclear import of the molecular chaperone Hsp70s is upregulated. Here, we identify a nuclear import pathway that operates during heat shock stress and is mediated by an evolutionarily conserved protein named "Hikeshi," which does not belong to the importin β family. Hikeshi binds to FG-Nups and translocates through nuclear pores on its own, showing characteristic features of nuclear transport carriers. In reconstituted transport, Hikeshi supports the nuclear import of the ATP form of Hsp70s, but not the ADP form, indicating the importance of the Hsp70 ATPase cycle in the import cycle. In living cells, depletion of Hikeshi inhibits heat shock-induced nuclear import of Hsp70s, reduces cell viability after heat shock stress, and significantly delays the attenuation and reversion of multiple heat shock-induced nuclear phenotypes. Nuclear Hsp70s rescue the effect of Hikeshi depletion at least in part. Thus, Hsp70s counteract heat shock-induced damage by acting inside of the nucleus.     

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.     

B23 regulates GADD45a nuclear translocation and contributes to GADD45a-induced cell cycle G2-M arrest

Gadd45a is an important player in cell cycle G2-M arrest in response to genotoxic stress. However, the underlying mechanism(s) by which Gadd45a exerts its role in the control of cell cycle progression remains to be further defined. Gadd45a interacts with Cdc2, dissociates the Cdc2-cyclin B1 complex, alters cyclin B1 nuclear localization, and thus inhibits the activity of Cdc2/cyclin B1 kinase. These observations indicate that Gadd45a nuclear translocation is closely associated with its role in cell cycle G2-M arrest. Gadd45a has been characterized as a nuclear protein, but it does not contain a classical nuclear localization signal, suggesting that Gadd45a nuclear translocation might be mediated through different nuclear import machinery. Here we show that Gadd45a associates directly with B23 (nucleophosmin), and the B23-interacting domain is mapped at the central region (61-100 amino acids) of the Gadd45a protein using a series of Myc tag-Gadd45a deletion mutants. Deletion of this central region disrupts Gadd45a association with B23 and abolishes Gadd45a nuclear translocation. Suppression of endogenous B23 through a short interfering RNA approach disrupts Gadd45a nuclear translocation and results in impaired Gadd45a-induced cell cycle G2-M arrest. These findings demonstrate a novel association of B23 and Gadd45a and implicate B23 as an important regulator in Gadd45a nuclear import.     

Troglitazone inhibits growth of MCF-7 breast carcinoma cells by targeting G1 cell cycle regulators

Peroxisome proliferator activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily. Ligand activation of PPARgamma has been shown to cause growth arrest in several human tumor cell types, but the underlying molecular mechanism has not been elucidated. We report here that the PPARgamma ligand troglitazone (TRO) inhibited MCF-7 cell proliferation by blocking events critical for G1 --> S progression. Flow cytometry demonstrated that TRO at 20 microM increased the percentage of cells in G1 from 51 to 69% after 24 h. Accumulation of cells in G1 was accompanied by an attenuation of Rb protein phosphorylation associated with decreased CDK4 and CDK2 activities. Inhibition of CDK activity by TRO correlates with decreased protein levels for several G1 regulators of Rb phosphorylation (cyclin D1, and CDKs 2, 4, and 6). Overexpression of cyclin D1 partially rescued MCF-7 cells from TRO-mediated G1 arrest. Targeting of G1 regulatory proteins, particularly cyclin D1, and the resulting induction of G1 arrest by TRO may provide a novel antiproliferative therapy for human breast cancer.     

Reevaluation of the role of the Pam18:Pam16 interaction in translocation of proteins by the mitochondrial Hsp70-based import motor

The heat-shock protein 70 (Hsp70)-based import motor, associated with the translocon on the matrix side of the mitochondrial inner membrane, drives translocation of proteins via cycles of binding and release. Stimulation of Hsp70's ATPase activity by the translocon-associated J-protein Pam18 is critical for this process. Pam18 forms a heterodimer with the structurally related protein Pam16, via their J-type domains. This interaction has been proposed to perform a critical regulatory function, inhibiting the ATPase stimulatory activity of Pam18. Using biochemical and genetic assays, we tested this hypothesis by assessing the in vivo function of Pam18 variants having altered abilities to stimulate Hsp70's ATPase activity. The observed pattern of genetic interactions was opposite from that predicted if the heterodimer serves an inhibitory function; instead the pattern was consistent with that of mutations known to cause reduction in the stability of the heterodimer. Analysis of a previously uncharacterized region of Pam16 revealed its requirement for formation of an active Pam18:Pam16 complex able to stimulate Hsp70's ATPase activity. Together, our data are consistent with the idea that Pam18 and Pam16 form a stable heterodimer and that the critical role of the Pam18:Pam16 interaction is the physical tethering of Pam18 to the translocon via its interaction with Pam16.     

Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16ink4.

The cell cycle in mammalian cells is regulated by a series of cyclins and cyclin-dependent kinases (CDKs). The G1/S checkpoint is mainly dictated by the kinase activities of the cyclin D-CDK4 and/or cyclin D-CDK6 complex and the cyclin E-CDK2 complex. These G1 kinases can in turn be regulated by cell cycle inhibitors, which may cause the cells to arrest at the G1 phase. In T-cell hybridomas, addition of anti-T-cell receptor antibody results not only in G1 arrest but also in apoptosis. In searching for a protein(s) which might interact with Nur77, an orphan steroid receptor required for activation-induced apoptosis of T-cell hybridomas, we have cloned a novel human and mouse CDK inhibitor, p19. The deduced p19 amino acid sequence consists of four ankyrin repeats with 48% identity to p16. The human p19 gene is located on chromosome 19p13, distinct from the positions of p18, p16, and p15. Its mRNA is expressed in all cell types examined. The p19 fusion protein can associate in vitro with CDK4 but not with CDK2, CDC2, or cyclin A, B, E, or D1 to D3. Addition of p19 protein can lead to inhibition of the in vitro kinase activity of cyclin D-CDK4 but not that of cyclin E-CDK2. In T-cell hybridoma DO11.10, p19 was found in association with CDK4 and CDK6 in vivo, although its association with Nur77 is not clear at this point. Thus, p19 is a novel CDK inhibitor which may play a role in the cell cycle regulation of T cells.     

Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1

Activation of stress-activated protein kinases (SAPKs) is essential for proper cell adaptation to extracellular stimuli. The exposure of yeast cells to high osmolarity, or mutations that lead to activation of the Hog1 SAPK, result in cell-cycle arrest. The mechanisms by which Hog1 and SAPKs in general regulate cell-cycle progression are not completely understood. Here we show that Hog1 regulates cell cycle progression at the G1 phase by a dual mechanism that involves downregulation of cyclin expression and direct targeting of the CDK-inhibitor protein Sic1. Hog1 interacts physically with Sic1 in vivo and in vitro, and phosphorylates a single residue at the carboxyl terminus of Sic1, which, in combination with the downregulation of cyclin expression, results in Sic1 stabilization and inhibition of cell-cycle progression. Cells lacking Sic1 or containing a Sic1 allele mutated in the Hog1 phosphorylation site are unable to arrest at G1 phase after Hog1 activation, and become sensitive to osmostress. Together, our data indicate that the Sic1 CDK-inhibitor is the molecular target for the SAPK Hog1 that is required to modulate cell-cycle progression in response to stress.     

Identification of a cdk-activating kinase in fission yeast.

We have identified a second cyclin-dependent kinase (cdk) in fission yeast, crk1, which encodes a 335 amino acid protein that is most closely related to the KIN28 gene product from Saccharomyces cerevisiae and to a cdk activating kinase (CAK) encoded by the MO15 gene from Xenopus laevis, crk1 is essential for viability and delta crk1 cells arrest with septa and condensed chromatin. We show that Crk1 associates with the Mcs2 mitotic catastrophe suppressor, a cyclin H-like molecule, and overexpression of crk1 rescues the cell-cycle arrest defect of a mcs2-75 cdc2-3w cdc25-22 triple mutant at high temperature. The Crk1-Mcs2 complex possesses CAK activity in vitro in that it phosphorylates human Cdk2 on Thr160 which results in its activation in the presence of cyclin A. In addition Crk1-Mcs2 effectively phosphorylates a peptide corresponding to the C-terminal repeat domain (CTD) of RNA polymerase II. We demonstrate that crk1 is allelic to the mcs6 mitotic catastrophe suppressor and that the X.laevis MO15 gene rescues the cell-cycle arrest of an mcs6-13 cdc2-3w cdc25-22 at high temperature. Together these data suggest that the Crk1-Mcs2 complex is a CAK that interacts genetically with Cdc2 in fission yeast.