Telomerase Inhibitor PinX1 Provides a Link between TRF1 and Telomerase to Prevent Telomere Elongation

The telomere is a functional chromatin structure that consists of G-rich repetitive sequences and various associated proteins. Telomeres protect chromosomal ends from degradation, provide escape from the DNA damage response, and regulate telomere lengthening by telomerase. Multiple proteins that localize at telomeres form a complex called shelterin/telosome. One component, TRF1, is a double-stranded telomeric DNA binding protein. Inactivation of TRF1 disrupts telomeric localization of other shelterin components and induces chromosomal instability. Here, we examined how the telomeric localization of shelterin components is crucial for TRF1-mediated telomere-associated functions. We found that many of the mTRF1 deficient phenotypes, including chromosomal instability, growth defects, and dysfunctional telomere damage response, were suppressed by the telomere localization of shelterin components in the absence of functional mTRF1. However, abnormal telomere signals and telomere elongation phenotypes were either not rescued or only partially rescued, respectively. These data suggest that TRF1 regulates telomere length and function by at least two mechanisms; in one TRF1 acts through the recruiting/tethering of other shelterin components to telomeres, and in the other TRF1 seems to play a more direct role.

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

During apoptosis, Smac (second mitochondria-derived activator of caspases)/DIABLO, an IAP (inhibitor of apoptosis protein)-binding protein, is released from mitochondria and potentiates apoptosis by relieving IAP inhibition of caspases. We demonstrate that exposure of MCF-7 cells to the death-inducing ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), results in rapid Smac release from mitochondria, which occurs before or in parallel with loss of cytochrome c. Smac release is inhibited by Bcl-2/Bcl-xL or by a pan-caspase inhibitor demonstrating that this event is caspase-dependent and modulated by Bcl-2 family members. Following release, Smac is rapidly degraded by the proteasome, an effect suppressed by co-treatment with a proteasome inhibitor. As the RING finger domain of XIAP possesses ubiquitin-protein ligase activity and XIAP binds tightly to mature Smac, an in vitro ubiquitination assay was performed which revealed that XIAP functions as a ubiquitin-protein ligase (E3) in the ubiquitination of Smac. Both the association of XIAP with Smac and the RING finger domain of XIAP are essential for ubiquitination, suggesting that the ubiquitin-protein ligase activity of XIAP may promote the rapid degradation of mitochondrial-released Smac. Thus, in addition to its well characterized role in inhibiting caspase activity, XIAP may also protect cells from inadvertent mitochondrial damage by targeting pro-apoptotic molecules for proteasomal degradation.

Telomere maintenance is essential for continued cell proliferation and chromosome stability. Telomeres are maintained by telomerase and a collection of associated proteins. The telomeric protein telomeric repeat binding factor 1 (TRF1) negatively regulates telomere length by inhibiting access of telomerase at telomere termini. Here we report that TRF1 interacts with the beta subunit of casein kinase 2 (CK2) and serves as a substrate for CK2. CK2-mediated phosphorylation is required for the efficient telomere binding of TRF1 in vitro and in vivo. Inhibition of CK2 by the CK2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole decreased the ability of TRF1 to bind telomeric DNA. The resulting telomere-unbound form of TRF1 was then ubiquitinated and degraded by the proteasome. Partial knockdown of CK2 by small interfering RNA resulted in removal of TRF1 from telomeres and subsequent degradation of TRF1. Mapping of the CK2 target site identified threonine 122 as a substrate in TRF1. A threonine to alanine change at this position led to a diminished DNA binding due to reduced dimerization of TRF1. In addition, phosphorylation of threonine 122 seemed critical for TRF1-mediated telomere length control. Our findings suggest that CK2-mediated phosphorylation of TRF1 plays an important role in modulating telomere length homeostasis by determining the levels of TRF1 at telomeres.

Biochemical and genetic findings accumulated over the past decade have established that the condensation of eukaryotic DNA in chromatin functions not only to constrain the genome within the boundaries of the cell nucleus but also to suppress gene activity in a general manner. This genetic repression extends from the level of the nucleosome, the primary unit of chromatin organization, where coiling of DNA on the surface of the nucleosome core particle impedes access to the transcriptional apparatus, to the higher order folding of nucleosome arrays and the organization of silent regions of chromatin (for reviews see Refs. 1van Holde K. Zlatanova J. Arents G. Moudrianakis E. Elgin S.C.R. Chromatin Structure and Gene Expression. Oxford University Press, Oxford, UK1995: 1-26Google Scholar, 2Ramakrishnan V. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 83-112Crossref PubMed Scopus (132) Google Scholar, 3Pruss D. Hayes J.J. Wolffe A.P. Bioessays. 1995; 17: 161-170Crossref PubMed Google Scholar, 4Grunstein M. Annu. Rev. Cell Biol. 1990; 6: 643-678Crossref PubMed Google Scholar, 5Kornberg R.D. Lorch Y. Annu. Rev. Cell Biol. 1992; 8: 563-589Crossref PubMed Google Scholar, 6Fletcher T.M. Hansen J.C. Crit. Rev. Eukaryotic Gene Expression. 1996; 6: 149-188Crossref PubMed Google Scholar and 105Koshland D. Strunnikov A. Annu. Rev. Cell Biol. 1996; 12: 305-333Crossref Scopus (283) Google Scholar). Chromatin structure is inextricably linked to transcriptional regulation, and recent studies show how chromatin is perturbed so as to facilitate transcription (for reviews see Refs. 7Adams C.C. Workman J.L. Cell. 1993; 72: 305-308Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 8Paranjape S.M. Kamakaka R.T. Kadonaga J.T. Annu. Rev. Biochem. 1994; 63: 265-297Crossref PubMed Google Scholar, 9Kornberg R.D. Lorch Y. Curr. Opin. Cell Biol. 1995; 7: 371-375Crossref PubMed Scopus (95) Google Scholar, 10Felsenfeld G. Cell. 1996; 86: 13-19Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 11Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (438) Google Scholar, 12Krude T. Elgin S.C.R. Curr. Biol. 1996; 6: 511-515Abstract Full Text Full Text PDF PubMed Google Scholar). Here, we review the substantial advances in the identification of histone acetyltransferases and histone deacetylases, whose opposing activities establish the steady-state level of histone acetylation, and progress in studies of multicomponent systems that require energy for the process of nucleosome disruption.Histone AcetylationSince the early discovery of histone acetylation by Allfrey and colleagues (13Allfrey V.G. Faulkner R. Mirsky A.E. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Google Scholar), this post-translation modification has been correlated with the processes of transcription and chromatin assembly. Acetylation occurs at specific lysines in the flexible N-terminal histone tails that protrude from the nucleosome surface (11Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (438) Google Scholar, 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar). Hyperacetylation of histones is associated with transcriptional activity or the potential for activity, whereas histone hypoacetylation is correlated with transcriptionally silent chromatin and heterochromatin. Histone acetylation is also associated with the active deposition and maturation of newly assembled nucleosomes during DNA replication (for reviews see Refs. 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar and 15Loidl P. Chromosoma. 1994; 103: 441-449Crossref PubMed Google Scholar). Acetylation reduces the net positive charge of the histones and weakens interactions with DNA (16Hong L. Schroth G.P. Matthews H.R. Yau P. Bradbury E.M. J. Biol. Chem. 1993; 268: 305-314Abstract Full Text PDF PubMed Google Scholar), inhibits the higher order folding of nucleosome arrays (17Hansen J.C. Wolffe A.P. Biochemistry. 1992; 31: 7977-7988Crossref PubMed Google Scholar, 18Garcia-Ramirez M. Rocchini C. Ausio J. J. Biol. Chem. 1995; 270: 17923-17928Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), and disrupts specific interactions with nonhistone regulators, as shown for the yeast silencer and repressor proteins Sir3 and Sir4 (19Thompson J.S. Ling X. Grunstein M. Nature. 1994; 369: 245-247Crossref PubMed Scopus (202) Google Scholar, 20Hecht A. Laroche T. Strahl-Bosinger S. Gasser S.M. Grunstein M. Cell. 1995; 80: 583-592Abstract Full Text PDF PubMed Scopus (674) Google Scholar) and Tup-1 (21Edmondson D.G. Smith M.M. Roth S.Y. Genes Dev. 1996; 10: 1247-1259Crossref PubMed Google Scholar).Tetrahymena Histone Acetyltransferase A and Yeast Gcn5In a convergence of biochemical and genetic studies, cloning of the p55 catalytic subunit of Tetrahymena nuclear (A-type) histone acetyltransferase (HAT) 1The abbreviations used are: HAT, histone acetyltransferase; P/CAF, p300/CBP-associated factor; CBP, CREB-binding protein; CREB, cyclic AMP response element binding protein; SAS, something about silencing; MOZ, monocytic leukemia zinc finger; MOF, males absent on the first; TAF, TBP-associated factor; TBP, TATA-binding protein; HDAC, histone deacetylase. revealed substantial sequence identity with yeast Gcn5, previously defined genetically as a transcriptional coactivator (22Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1210) Google Scholar). 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However, unlike the native HAT A enzyme, recombinant Gcn5 cannot acetylate nucleosomal histones, implying that other subunits in the complex must influence its activity on chromatin. Genetic and biochemical studies reveal at least two interacting proteins, Ada2 and Ada3, that form a complex with Gcn5 (23Candau R. Zhou J.X. Allis C.D. Berger S.L. EMBO J. 1997; 16: 555-565Crossref PubMed Scopus (174) Google Scholar,27Marcus G.A. Silverman N. Berger S.L. Horiuchi J. Guarente L. EMBO J. 1994; 13: 4807-4815Crossref PubMed Scopus (234) Google Scholar, 28Horiuchi J. Silverman N. Marcus G.A. Guarente L. Mol. Cell. Biol. 1995; 15: 1203-1209Crossref PubMed Google Scholar, 29Georgakopoulos T. Gounalaki N. Thireos G. Mol. Gen. Genet. 1995; 246: 723-728Crossref PubMed Scopus (53) Google Scholar). Binding of Ada2 to the activation domains of the transcriptional activators VP16 and Gcn4 in vitro suggests a mechanism by which promoter targeting of Gcn5 might be achieved (30Silverman N. Agapite J. Guarente L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11665-11668Crossref PubMed Scopus (96) Google Scholar,31Barlev N.A. Candau R. Wang L. Darpino P. Silverman N. Berger S.L. J. Biol. Chem. 1995; 270: 19337-19344Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar).P/CAF, p300/CBP, SAS, MOZ, and MOFAn increasing number of putative or demonstrated histone acetyltransferases have emerged in the past year. P/CAF (p300/CBP-associated factor) is a novel histone acetyltransferase isolated on the basis of sequence similarity to human and yeast Gcn5 (32Yang X.-J. Ogryzko V.V. Nishizawa J. Howard B.H. Nakayani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1283) Google Scholar), which interacts with the highly related transcriptional coactivators p300/CBP. Like Gcn5, recombinant P/CAF has intrinsic HAT activity for free histones H3 and H4, but unlike Gcn5, P/CAF is also able to acetylate nucleosomal histone H3. p300/CBP (CREB-binding protein) is itself a histone acetyltransferase with no resemblance in sequence to the other acetyltransferases (33Ogryzko V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2274) Google Scholar, 106Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1480) Google Scholar). The bacterially expressed p300/CBP protein is unique among HAT polypeptides in that it can acetylate all four core histones free in solution or when complexed in the nucleosome; acetylation on histone H4 occurs at lysines 5, 8, 12, and 16, the same positions that are subject to acetylation in vivo (32Yang X.-J. Ogryzko V.V. Nishizawa J. Howard B.H. Nakayani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1283) Google Scholar). p300 and CBP are known to physically interact with numerous transcription factors activated by signaling cascades, including CREB, c-Jun/v-Jun, Fos, and nuclear hormone receptors, and are also targets for the E1A oncoprotein (for a review see Ref. 34Janknecht R. Hunter T. Nature. 1996; 383: 22-23Crossref PubMed Scopus (337) Google Scholar). Whether histones are substrates of p300 in vivo remains to be determined.The yeast SAS, human MOZ and Tip60, and fly MOF proteins constitute a different class of putative acetyltransferases, characterized by a ∼300-amino acid region of significant similarity that contains a C2CH zinc finger motif and a subregion similar to HATs and other acetyltransferases (35Reifsnyder C. Lowell J. Clarke A. Pillus L. Nat. Genet. 1996; 14: 42-49Crossref PubMed Scopus (234) Google Scholar, 36Borrow J. Stanton Jr., V.P. Andresen J.M. Becher R. Behm F.G. Chaganti R.S.K. Civin C.I. Disteche C. 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Frischauf A.M. Horsman D. Mitelman F. Volinia S. Watmore A.E. Housman D.E. Nat. Genet. 1996; 14: 33-41Crossref PubMed Scopus (609) Google Scholar).TAF250TFIID, the general transcription factor complex of TBP (TATA-binding protein) and the associated TAF proteins, has been found to contain a HAT activity. Unique among the various TAFs, TAFII250 alone or in the TFIID complex has both a serine kinase activity selective for RAP74 (a subunit of TFIIF) and a HAT activity (39Dikstein R. Ruppert S. Tjian R. Cell. 1996; 84: 781-790Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 40Mizzen C.A. Yang X.-J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J.L. Berger S.L. Kouzarides T. Nakatani Y. Aliis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). Like Gcn5, TAFII250 preferentially acetylates free histone H3 over H4 and has little or no activity on nucleosomal histones (40Mizzen C.A. Yang X.-J. Kokubo T. 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These findings may perhaps be related to the acetylation of histone H4 at lysine 12, which is required for transcriptional silencing in yeast (50Braunstein M. Sorbel R.E. Allis C.D. Turner B.M. Broach J.R. Mol. Cell. Biol. 1996; 16: 4349-4356Crossref PubMed Google Scholar) and which is also associated with heterochromatin in Drosophila (51Turner B.M. Birley A.J. Lavender J. Cell. 1992; 69: 375-384Abstract Full Text PDF PubMed Google Scholar), despite net hypoacetylation.More in line with the anticipated involvement of histone deacetylases in transcriptional repression is the physical association of the heteromeric Mad (Mxi1)/Max DNA binding repressors involved in controlling cell proliferation and differentiation with mammalian RPD3 homologs and with Sin3, a conserved transcriptional co-repressor genetically linked to yeast Rpd3 (45Hassig C.A. Fleischer T.C. Billin A.N. Schreibert S.L. Ayer D.E. 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Cell. 1997; 89: Full Text Full Text PDF PubMed Scopus Google Scholar). of genes by the yeast protein also of a complex Sin3 and Rpd3 D. Struhl K. Cell. 1997; 89: Full Text Full Text PDF PubMed Scopus Google Scholar). This of strong for a of repression by the of histone deacetylase complexes to the of so of chromatin structure a of that for is the of nucleosomal histones during the histones or from and how might a structure interactions between DNA binding S. M. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google nucleosomes that are or to the structure of DNA have different for in chromatin are by proteins as and Burley S.K. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and by the complex itself L. A. D. M. Cell. 1997; 89: Full Text Full Text PDF PubMed Google chromatin are for a gene and at which of the activation the binding of regulators, of the or activities to specific chromosomal or they the chromatin of for other enzymes are by is the of the modification a nucleosome and how is the modification the activities of the histone acetyltransferases and histone deacetylases with the family of nucleosome on higher order chromatin or is it by higher order chromatin to these and other not only for the of gene but also for of Biochemical and genetic findings accumulated over the past decade have established that the condensation of eukaryotic DNA in chromatin functions not only to constrain the genome within the boundaries of the cell nucleus but also to suppress gene activity in a general manner. This genetic repression extends from the level of the nucleosome, the primary unit of chromatin organization, where coiling of DNA on the surface of the nucleosome core particle impedes access to the transcriptional apparatus, to the higher order folding of nucleosome arrays and the organization of silent regions of chromatin (for reviews see Refs. 1van Holde K. Zlatanova J. Arents G. Moudrianakis E. Elgin S.C.R. Chromatin Structure and Gene Expression. Oxford University Press, Oxford, UK1995: 1-26Google Scholar, 2Ramakrishnan V. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 83-112Crossref PubMed Scopus (132) Google Scholar, 3Pruss D. Hayes J.J. Wolffe A.P. Bioessays. 1995; 17: 161-170Crossref PubMed Google Scholar, 4Grunstein M. Annu. Rev. Cell Biol. 1990; 6: 643-678Crossref PubMed Google Scholar, 5Kornberg R.D. Lorch Y. Annu. Rev. Cell Biol. 1992; 8: 563-589Crossref PubMed Google Scholar, 6Fletcher T.M. Hansen J.C. Crit. Rev. Eukaryotic Gene Expression. 1996; 6: 149-188Crossref PubMed Google Scholar and 105Koshland D. Strunnikov A. Annu. Rev. Cell Biol. 1996; 12: 305-333Crossref Scopus (283) Google Scholar). Chromatin structure is inextricably linked to transcriptional regulation, and recent studies show how chromatin is perturbed so as to facilitate transcription (for reviews see Refs. 7Adams C.C. Workman J.L. Cell. 1993; 72: 305-308Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 8Paranjape S.M. Kamakaka R.T. Kadonaga J.T. Annu. Rev. Biochem. 1994; 63: 265-297Crossref PubMed Google Scholar, 9Kornberg R.D. Lorch Y. Curr. Opin. Cell Biol. 1995; 7: 371-375Crossref PubMed Scopus (95) Google Scholar, 10Felsenfeld G. Cell. 1996; 86: 13-19Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 11Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (438) Google Scholar, 12Krude T. Elgin S.C.R. Curr. Biol. 1996; 6: 511-515Abstract Full Text Full Text PDF PubMed Google Scholar). Here, we review the substantial advances in the identification of histone acetyltransferases and histone deacetylases, whose opposing activities establish the steady-state level of histone acetylation, and progress in studies of multicomponent systems that require energy for the process of nucleosome Histone AcetylationSince the early discovery of histone acetylation by Allfrey and colleagues (13Allfrey V.G. Faulkner R. Mirsky A.E. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Google Scholar), this post-translation modification has been correlated with the processes of transcription and chromatin assembly. Acetylation occurs at specific lysines in the flexible N-terminal histone tails that protrude from the nucleosome surface (11Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (438) Google Scholar, 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar). Hyperacetylation of histones is associated with transcriptional activity or the potential for activity, whereas histone hypoacetylation is correlated with transcriptionally silent chromatin and heterochromatin. Histone acetylation is also associated with the active deposition and maturation of newly assembled nucleosomes during DNA replication (for reviews see Refs. 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar and 15Loidl P. Chromosoma. 1994; 103: 441-449Crossref PubMed Google Scholar). Acetylation reduces the net positive charge of the histones and weakens interactions with DNA (16Hong L. Schroth G.P. Matthews H.R. Yau P. Bradbury E.M. J. Biol. Chem. 1993; 268: 305-314Abstract Full Text PDF PubMed Google Scholar), inhibits the higher order folding of nucleosome arrays (17Hansen J.C. Wolffe A.P. Biochemistry. 1992; 31: 7977-7988Crossref PubMed Google Scholar, 18Garcia-Ramirez M. Rocchini C. Ausio J. J. Biol. Chem. 1995; 270: 17923-17928Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), and disrupts specific interactions with nonhistone regulators, as shown for the yeast silencer and repressor proteins Sir3 and Sir4 (19Thompson J.S. Ling X. Grunstein M. Nature. 1994; 369: 245-247Crossref PubMed Scopus (202) Google Scholar, 20Hecht A. Laroche T. Strahl-Bosinger S. Gasser S.M. Grunstein M. Cell. 1995; 80: 583-592Abstract Full Text PDF PubMed Scopus (674) Google Scholar) and Tup-1 (21Edmondson D.G. Smith M.M. Roth S.Y. Genes Dev. 1996; 10: 1247-1259Crossref PubMed Google Scholar). the early discovery of histone acetylation by Allfrey and colleagues (13Allfrey V.G. Faulkner R. Mirsky A.E. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Google Scholar), this post-translation modification has been correlated with the processes of transcription and chromatin assembly. Acetylation occurs at specific lysines in the flexible N-terminal histone tails that protrude from the nucleosome surface (11Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (438) Google Scholar, 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar). Hyperacetylation of histones is associated with transcriptional activity or the potential for activity, whereas histone hypoacetylation is correlated with transcriptionally silent chromatin and heterochromatin. Histone acetylation is also associated with the active deposition and maturation of newly assembled nucleosomes during DNA replication (for reviews see Refs. 14Turner B.M. O'Neil L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Google Scholar and 15Loidl P. Chromosoma. 1994; 103: 441-449Crossref PubMed Google Scholar). Acetylation reduces the net positive charge of the histones and weakens interactions with DNA (16Hong L. Schroth G.P. Matthews H.R. Yau P. Bradbury E.M. J. Biol. Chem. 1993; 268: 305-314Abstract Full Text PDF PubMed Google Scholar), inhibits the higher order folding of nucleosome arrays (17Hansen J.C. Wolffe A.P. Biochemistry. 1992; 31: 7977-7988Crossref PubMed Google Scholar, 18Garcia-Ramirez M. Rocchini C. Ausio J. J. Biol. Chem. 1995; 270: 17923-17928Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), and disrupts specific interactions with nonhistone regulators, as shown for the yeast silencer and repressor proteins Sir3 and Sir4 (19Thompson J.S. Ling X. Grunstein M. Nature. 1994; 369: 245-247Crossref PubMed Scopus (202) Google Scholar, 20Hecht A. Laroche T. Strahl-Bosinger S. Gasser S.M. Grunstein M. Cell. 1995; 80: 583-592Abstract Full Text PDF PubMed Scopus (674) Google Scholar) and Tup-1 (21Edmondson D.G. Smith M.M. Roth S.Y. Genes Dev. 1996; 10: 1247-1259Crossref PubMed Google Scholar). Tetrahymena Histone Acetyltransferase A and Yeast Gcn5In a convergence of biochemical and genetic studies, cloning of the p55 catalytic subunit of Tetrahymena nuclear (A-type) histone acetyltransferase (HAT) 1The abbreviations used are: HAT, histone acetyltransferase; P/CAF, p300/CBP-associated factor; CBP, CREB-binding protein; CREB, cyclic AMP response element binding protein; SAS, something about silencing; MOZ, monocytic leukemia zinc finger; MOF, males absent on the first; TAF, TBP-associated factor; TBP, TATA-binding protein; HDAC, histone deacetylase. revealed substantial sequence identity with yeast Gcn5, previously defined genetically as a transcriptional coactivator (22Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1210) Google Scholar). The catalytic domain of the Gcn5 HAT is required for coactivator function in vivo, providing a genetic link between histone modification and transcriptional activation (23Candau R. Zhou J.X. Allis C.D. Berger S.L. EMBO J. 1997; 16: 555-565Crossref PubMed Scopus (174) Google Scholar). As a human GCN5 homolog has been identified, this HAT is likely to be widely conserved (24Candau R. Moore P.A. Wang L. Barlev N. Ying C.Y. Rosen C.A. Berger S.L. Mol. Cell. Biol. 1996; 16: 593-602Crossref PubMed Scopus (154) Google Scholar, 25Wang L. Mizzen C. Ying C. Candau R. Barlev N. Brownell J. Allis C.D. Berger S.L. Mol. Cell. Biol. 1997; 17: 519-527Crossref PubMed Google Scholar). Bacterially expressed yeast Gcn5 protein acetylates free histone H3 strongly at lysine 14 and histone H4 weakly at lysines 8 and 16 (26Kuo M.-H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1996; 383: 269-272Crossref PubMed Scopus (480) Google Scholar). However, unlike the native HAT A enzyme, recombinant Gcn5 cannot acetylate nucleosomal histones, implying that other subunits in the complex must influence its activity on chromatin. Genetic and biochemical studies reveal at least two interacting proteins, Ada2 and Ada3, that form a

The Xenopus cyclin-dependent kinase (CDK) inhibitor, p27(Xic1) (Xic1), binds to CDK2-cyclins and proliferating cell nuclear antigen (PCNA), inhibits DNA synthesis in Xenopus extracts, and is targeted for ubiquitin-mediated proteolysis. Previous studies suggest that Xic1 ubiquitination and degradation are coupled to the initiation of DNA replication, but the precise timing and molecular mechanism of Xic1 proteolysis has not been determined. Here we demonstrate that Xic1 proteolysis is temporally restricted to late replication initiation following the requirements for DNA polymerase alpha-primase, replication factor C, and PCNA. Our studies also indicate that Xic1 degradation is absolutely dependent upon the binding of Xic1 to PCNA in both Xenopus egg and gastrulation stage extracts. Additionally, extracts depleted of PCNA do not support Xic1 proteolysis. Importantly, while the addition of recombinant wild-type PCNA alone restores Xic1 degradation, the addition of a PCNA mutant defective for trimer formation does not restore Xic1 proteolysis in PCNA-depleted extracts, suggesting Xic1 proteolysis requires both PCNA binding to Xic1 and the ability of PCNA to be loaded onto primed DNA by replication factor C. Taken together, our studies suggest that Xic1 is targeted for ubiquitination and degradation during DNA polymerase switching through its interaction with PCNA at a site of initiation.

The Werner syndrome helicase (WRN) participates in DNA replication, double strand break repair, telomere maintenance, and p53 activation. Mutations of wrn cause Werner syndrome (WS), an autosomal recessive premature aging disorder associated with cancer predisposition, atherosclerosis, and other aging related symptoms. Here, we report that WRN is a novel cofactor for HIV-1 replication. Immortalized human WRN(-/-) WS fibroblasts, lacking a functional wrn gene, are impaired for basal and Tat-activated HIV-1 transcription. Overexpression of wild-type WRN transactivates the HIV-1 long terminal repeat (LTR) in the absence of Tat, and WRN cooperates with Tat to promote high-level LTR transactivation. Ectopic WRN induces HIV-1 p24(Gag) production and retroviral replication in HIV-1-infected H9(HIV-1IIIB) lymphocytes. A dominant-negative helicase-minus mutant, WRN(K577M), inhibits LTR transactivation and HIV-1 replication. Inhibition of endogenous WRN, through co-expression of WRN(K577M), diminishes recruitment of p300/CREB-binding protein-associated factor (PCAF) and positive transcription elongation factor b (P-TEFb) to Tat/transactivation response-RNA complexes, and immortalized WRN(-/-) WS fibroblasts exhibit comparable defects in recruitment of PCAF and P-TEFb to the HIV-1 LTR. Our results demonstrate that WRN is a novel cellular cofactor for HIV-1 replication and suggest that the WRN helicase participates in the recruitment of PCAF/P-TEFb-containing transcription complexes. WRN may be a plausible target for antiretroviral therapy.

Mcm2, a member of the Mcm2-7 protein family essential for the initiation of DNA replication, has several biochemical activities including the ability to inhibit the Mcm4,6,7 helicase. In this study, we characterized the activities associated with Mcm2 and determined the region required for them. It was found that Mcm2 deleted at an amino-terminal portion is able to bind to an Mcm4,6,7 hexameric complex and to inhibit its DNA helicase activity. The same deletion mutant of Mcm2 and the carboxyl-terminal half of Mcm2 were both able to bind to Mcm4, suggesting that the carboxyl-half of Mcm2 binds to Mcm4 to disassemble the Mcm4,6,7 hexamer. Phosphorylation of Mcm2,4,6,7 complexes with Cdc7 kinase showed that the amino-terminal region of Mcm2 is required for the phosphorylation, and it contains major Cdc7-mediated phosphorylation sites. We also found that Mcm2 itself can assemble a nucleosome-like structure in vitro in the presence of H3/H4 histones. The amino-terminal region of Mcm2 was required for the activity where a histone-binding domain is located. Finally, we identified a region required for the nuclear localization of Mcm2. The function of Mcm2 is discussed based on these biochemical characteristics.

Binding of nerve growth factor (NGF) to the p75 neurotrophin receptor (p75) in cultured hippocampal neurons has been reported to cause seemingly contrasting effects, namely ceramide-dependent axonal outgrowth of freshly plated neurons, versus Jun kinase (Jnk)-dependent cell death in older neurons. We now show that the apoptotic effects of NGF in hippocampal neurons are observed only from the 2nd day of culture onward. This switch in the effect of NGF is correlated with an increase in p75 expression levels and increasing levels of ceramide generation as the cultures mature. NGF application to neuronal cultures from p75(exonIII-/-) mice had no effect on ceramide levels and did not affect neuronal viability. The neutral sphingomyelinase inhibitor, scyphostatin, inhibited NGF-induced ceramide generation and neuronal death, whereas hippocampal neurons cultured from acid sphingomyelinase(-/-) mice were as susceptible to NGF-induced death as wild type neurons. The acid ceramidase inhibitor, (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, enhanced cell death, supporting a role for ceramide itself and not a downstream lipid metabolite. Finally, scyphostatin inhibited NGF-induced Jnk phosphorylation in hippocampal neurons. These data indicate an initiating role of ceramide generated by neutral sphingomyelinase in the diverse neuronal responses induced by binding of neurotrophins to p75.

Although several isoforms of protein kinase C (PKC) have been implicated in T lymphocyte activation events, little is known about their mode of action. To address the role of PKCzeta in T cell activation, we have generated Jurkat T cell transfectants expressing either the wild type (J-PKCzeta) or "kinase-dead" mutant (J-PKCzeta(mut)) versions of this protein. Expression of PKCzeta but not PKCzeta(mut) increased transcriptional activation mediated by the NF-kappaB or nuclear factor of activated T cells (NFAT). PKCzeta cooperates with calcium ionophore and with NFAT1 or NFAT2 proteins to enhance transcriptional activation of a NFAT reporter construct. However, neither NFAT nuclear translocation nor DNA binding were in J-PKCzeta cells. Our results show that PKCzeta enhanced transcriptional activity mediated by Gal4-NFAT1 fusion proteins containing the N-terminal transactivation domain of human NFAT1. Interestingly, PKCzeta synergizes with calcineurin to induce transcriptional activation driven by the NFAT1 transactivation domain. Co-precipitation experiments showed physical interaction between PKCzeta and NFAT1 or NFAT2 isoforms. Even more, PKCzeta was able to phosphorylate recombinant glutathione S-transferase-NFAT1 (1-385) protein. These data reveal a new role of PKCzeta in T cells through the control of NFAT function by modulating the activity of its transactivation domain.

Transforming growth factor beta (TGF-beta) and related growth factors are essential regulators of embryogenesis and tissue homeostasis. The signaling pathways mediated by their receptors and Smad proteins are precisely modulated by various means. Xenopus BAMBI (bone morphogenic protein (BMP) and activin membrane-bound inhibitor) has been shown to function as a general negative regulator of TGF-beta/BMP/activin signaling. Here, we provide evidence that human BAMBI (hBAMBI), like its Xenopus homolog, inhibits TGF-beta- and BMP-mediated transcriptional responses as well as TGF-beta-induced R-Smad phosphorylation and cell growth arrest, whereas knockdown of endogenous BAMBI enhances the TGF-beta-induced reporter expression. Mechanistically, in addition to interfering with the complex formation between the type I and type II receptors, hBAMBI cooperates with Smad7 to inhibit TGF-beta signaling. hBAMBI forms a ternary complex with Smad7 and the TGF-beta type I receptor ALK5/TbetaRI and inhibits the interaction between ALK5/TbetaRI and Smad3, thus impairing Smad3 activation. These findings provide a novel insight to understand the molecular mechanism underlying the inhibitory effect of BAMBI on TGF-beta signaling.

Ordered cell cycle progression requires the expression and activation of several cyclins and cyclin-dependent kinases (Cdks). Hyperosmotic stress causes growth arrest possibly via proteasome-mediated degradation of cyclin D1. We studied the effect of hyposmotic conditions on three colonic (Caco2, HRT18, HT29) and two pancreatic (AsPC-1 and PaCa-2) cell lines. Hyposmosis caused reversible cell growth arrest of the five cell lines in a cell cycle-independent fashion, although some cell lines accumulated at the G(1)/S interface. Growth arrest was followed by apoptosis or by formation of multinucleated giant cells, which is consistent with cell cycle catastrophe. Hyposmosis dramatically decreased Cdc2, Cdk2, Cdk4, cyclin B1, and cyclin D3 expression in a time-dependent fashion, in association with an overall decrease in cellular protein synthesis. However, some protein levels remained unaltered, including cyclin E and keratin 8. Selective proteasome inhibition prevented Cdk and cyclin degradation and reversed hyposmotic stress-induced growth arrest, whereas calpain and lysosome enzyme inhibitors had no measurable effect on cell cycle protein degradation. Therefore, hyposmotic stress inhibits cell growth and, depending on the cell type, causes cell cycle catastrophe with or without apoptosis. The growth arrest is due to decreased protein synthesis and proteasome activation, with subsequent degradation of several cyclins and Cdks.

DNA Damage Control by Novel DNA Polymerases: Translesion Replication and Mutagenesis

Hsp90 was originally identified as one of several conserved heat shock proteins. Like the other major classes of heat shock proteins, Hsp90 exhibits general protective chaperone properties, such as preventing the unspecific aggregation of non-native proteins (1Wiech H. Buchner J. Zimmermann R. Jakob U. Nature. 1992; 358: 169-170Crossref PubMed Scopus (417) Google Scholar). However, Hsp90 seems to be more selective than the other promiscuous general chaperones, as it preferentially interacts with a specific subset of the proteome (2Picard D. CMLS Cell. Mol. Life Sci. 2002; 59: 1640-1648Crossref PubMed Scopus (645) Google Scholar). Another specific feature of Hsp90 is its regulatory role of inducing conformational changes in folded, native-like substrate proteins that lead to their activation or stabilization (3Jakob U. Lilie H. Meyer I. Buchner J. J. Biol. Chem. 1995; 270: 7288-7294Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Recently, the three-dimensional structures of full-length Hsp90 from Escherichia coli, yeast, and the endoplasmic reticulum were solved (4Shiau A.K. Harris S.F. Southworth D.R. Agard D.A. Cell. 2006; 127: 329-340Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 5Ali M.M. Roe S.M. Vaughan C.K. Meyer P. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Nature. 2006; 440: 1013-1017Crossref PubMed Scopus (683) Google Scholar, 6Dollins D.E. Warren J.J. Immormino R.M. Gewirth D.T. Mol. Cell. 2007; 28: 41-56Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 7Pearl L.H. Prodromou C. Annu. Rev. Biochem. 2006; 75: 271-294Crossref PubMed Scopus (875) Google Scholar). Together with sequence data, these showed that, although Hsp90 maintained its general domain structure from bacteria to man, distinct changes seem to have adapted Hsp90 to the more complex protein environment of the eukaryotic cell. Concomitant with the occurrence of a long charged linker connecting the N- 3The abbreviations used are: N-domain, N-terminal domain; M-domain, middle domain; SHR, steroid hormone receptor; TPR, tetratricopeptide repeat; PPIase, peptidylprolyl cis/trans-isomerase; eNOS, epithelial nitric-oxide synthase. 3The abbreviations used are: N-domain, N-terminal domain; M-domain, middle domain; SHR, steroid hormone receptor; TPR, tetratricopeptide repeat; PPIase, peptidylprolyl cis/trans-isomerase; eNOS, epithelial nitric-oxide synthase. and M-domains, the eukaryotic protein exhibits an extension of the C-terminal domain, which includes the conserved amino acid motif MEEVD at the C terminus (8Chen S. Sullivan W.P. Toft D.O. Smith D.F. Cell Stress Chaperones. 1998; 3: 118-129Crossref PubMed Scopus (166) Google Scholar). This region serves as the major interaction site for a cohort of co-chaperones (Table 1) (9Richter K. Meinlschmidt B. Buchner J. Buchner J. Kiefhaber T Protein Folding Handbook. Wiley-VCH Verlag GmbH & Co., Weinheim, Germany2005: 768-829Crossref Scopus (1) Google Scholar), which apparently support Hsp90 in the folding and activation of its substrate proteins in eukaryotes. In this review, we summarize the current knowledge on the functional principles of this molecular machine, including the ATP-driven chaperone cycle of Hsp90 and its regulation by co-chaperones and post-translational modifications.TABLE 1Selected Hsp90 cofactors Open table in a new tab Hsp90 is a flexible dimer. Each monomer consists of three domains: the N-domain, connected by a long linker sequence (in eukaryotes) to an M-domain, which is followed by a C-terminal dimerization domain (Fig. 1). The N-domain possesses a deep ATP-binding pocket (10Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar), where ATP is bound in an unusual kinked manner. ATP hydrolysis by Hsp90 is rather slow: Hsp90 from yeast hydrolyzes one molecule of ATP every 1 or 2 min (11Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (610) Google Scholar, 12Scheibel T. Weikl T. Buchner J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1495-1499Crossref PubMed Scopus (229) Google Scholar), and human Hsp90 hydrolyzes one molecule of ATP every 20 min (0.04 min–1) (13McLaughlin S.H. Smith H.W. Jackson S.E. J. Mol. Biol. 2002; 315: 787-798Crossref PubMed Scopus (210) Google Scholar). The ATPase activity is essential for the function of Hsp90 in yeast (11Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (610) Google Scholar, 14Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (479) Google Scholar). The slow hydrolysis suggests that complex conformational rearrangements of Hsp90 are coupled to the ATPase reaction and that these represent the rate-limiting step of the enzyme. The first steps of these conformational changes were elucidated recently in detail (15Richter K. Moser S. Hagn F. Friedrich R. Hainzl O. Heller M. Schlee S. Kessler H. Reinstein J. Buchner J. J. Biol. Chem. 2006; 281: 11301-11311Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar): upon ATP binding, a short segment of the N-domain called the “ATP lid” changes its position and flaps over the binding pocket (Fig. 1, steps 2 and 3). This releases a short N-terminal segment from its original position (16Richter K. Reinstein J. Buchner J. J. Biol. Chem. 2002; 277: 44905-44910Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In a subsequent reaction, this segment binds to the respective N-domain of the other subunit in the dimer, producing a strand-swapped, transiently dimerized N-terminal conformation (step 3) (5Ali M.M. Roe S.M. Vaughan C.K. Meyer P. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Nature. 2006; 440: 1013-1017Crossref PubMed Scopus (683) Google Scholar, 15Richter K. Moser S. Hagn F. Friedrich R. Hainzl O. Heller M. Schlee S. Kessler H. Reinstein J. Buchner J. J. Biol. Chem. 2006; 281: 11301-11311Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). These N-terminal rearrangements result in further conformational changes throughout the entire Hsp90 dimer leading to a twisted and compacted dimer, in which N- and M-domains associate and the distance between M-domains is shortened by 40 Å (5Ali M.M. Roe S.M. Vaughan C.K. Meyer P. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Nature. 2006; 440: 1013-1017Crossref PubMed Scopus (683) Google Scholar). The association of N- and M-domains completes the active site of this “split ATPase” (step 4). Recently, a similar progression of steps was shown to occur also for the endoplasmic homolog Grp94 (17Frey S. Leskovar A. Reinstein J. Buchner J. J. Biol. Chem. 2007; 282: 35612-35620Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), mitochondrial TRAP1 (6Dollins D.E. Warren J.J. Immormino R.M. Gewirth D.T. Mol. Cell. 2007; 28: 41-56Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 18Leskovar, A., Wegele, H., Werbeck, N. D., Buchner, J., and Reinstein, J. (2008) 283, 11677–11688Google Scholar), and human Hsp90 (19Richter K. Soroka J. Skalniak L. Leskovar A. Hessling M. Reinstein J. Buchner J. J. Biol. Chem. 2008; 283: 17757-17765Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Therefore, the scenario outlined above seems to be the ubiquitously conserved ATPase mechanism for Hsp90. Interestingly, the unusual way in which ATP is bound by Hsp90 is perfectly mimicked by some natural compounds, such as geldanamycin and radicicol. These are highly specific and potent inhibitors of the Hsp90 ATPase (20Roe S.M. Prodromou C. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. J. Med. Chem. 1999; 42: 260-266Crossref PubMed Scopus (871) Google Scholar), blocking the maturation of substrate proteins and eventually resulting in their degradation (21Whitesell L. Mimnaugh E.G. De Costa B. Myers C.E. Neckers L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8324-8328Crossref PubMed Scopus (1310) Google Scholar). As several Hsp90 substrate proteins are kinases, which can be deregulated in the development of cancer, derivatives of Hsp90 inhibitors are currently being investigated as anticancer therapeutics at the stage of clinical trials (22Sharp S. Workman P. Adv. Cancer Res. 2006; 95: 323-348Crossref PubMed Scopus (269) Google Scholar). Current models assume that the conformational changes associated with ATP hydrolysis are required for reaching or maintaining an activated state of a substrate protein. In well studied examples such as the SHRs, several cofactors interact with Hsp90 in a sequential manner to assemble a functional chaperone machinery (23Pratt W.B. Toft D.O. Exp. Biol. Med. (Maywood). 2003; 228: 111-133Crossref PubMed Scopus (1239) Google Scholar, 24Smith D.F. Mol. Endocrinol. 1993; 7: 1418-1429Crossref PubMed Scopus (251) Google Scholar). The basis for this ordered succession of different assemblies can now be rationalized, as it turned out that several Hsp90 cofactors display a strong binding preference for specific Hsp90 conformations. The loading of an SHR onto Hsp90 requires the cooperation of Hsp90 with the chaperone Hsp70 and its cofactor Hsp40 (25Smith D.F. Sullivan W.P. Marion T.N. Zaitsu K. Madden B. McCormick D.J. Toft D.O. Mol. Cell. Biol. 1993; 13: 869-876Crossref PubMed Scopus (246) Google Scholar). Moreover, both chaperones become physically linked by an adaptor protein called Hop/Sti1 (Table 1). This co-chaperone binds via small helical TPR domains to the C-terminal ends of Hsp70 and Hsp90 (26Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (991) Google Scholar). It seems that Hsp70 stabilizes the SHR in a conformation that can be recognized and bound by Hsp90. However, experimental evidence for this notion is largely lacking. How the substrate in this complex is transferred from Hsp70 to Hsp90 is also still unclear. It might be that the bridging by Hop/Sti1 selects for Hsp90 molecules in a conformation competent for substrate binding in addition to increasing the local concentration of Hsp70 and Hsp90. For the progression of the chaperone cycle, empty Hsp70 and Hop/Sti1 have to dissociate, and other co-chaperones such as specific PPIases and p23/Sba1 enter the complex (Table 1) (24Smith D.F. Mol. Endocrinol. 1993; 7: 1418-1429Crossref PubMed Scopus (251) Google Scholar). These PPIases also possess a TPR domain, which binds to the C-terminal end of Hsp90. The second cofactor, p23/Sba1, associates with the N-terminally dimerized conformation of Hsp90 (27Prodromou C. Panaretou B. Chohan S. Siligardi G. O'Brien R. Ladbury J.E. Roe S.M. Piper P.W. Pearl L.H. EMBO J. 2000; 19: 4383-4392Crossref PubMed Google Scholar, 28Richter K. Walter S. Buchner J. J. Mol. Biol. 2004; 342: 1403-1413Crossref PubMed Scopus (119) Google Scholar), making it likely that the dramatic conformational rearrangement from the open to the closed state of Hsp90 occurs at this stage of the Hsp90 chaperone cycle (Fig. 1, steps 3 and 4). This closed conformation is metastable and upon ATP hydrolysis returns to the open state (Fig. 1, step 5) (28Richter K. Walter S. Buchner J. J. Mol. Biol. 2004; 342: 1403-1413Crossref PubMed Scopus (119) Google Scholar). The bound substrate protein dissociates in turn from Hsp90, permitting a new round of the cycle. The first steps of the cycle for the maturation of signaling kinases is a variation of the scheme described above. Here, the kinase-specific Hsp90 cofactor Cdc37 seems to associate with substrate kinases in their inactive forms first. This complex may then be loaded onto Hsp90 (29Kimura Y. Rutherford S.L. Miyata Y. Yahara I. Freeman B.C. Yue L. Morimoto R.I. Lindquist S. Genes Dev. 1997; 11: 1775-1785Crossref PubMed Scopus (175) Google Scholar). The following steps are less clear yet. It may be that also for kinases, Hsp70, Hsp40, and Hop/Sti1 are additionally required (30Caplan A.J. Mandal A.K. Theodoraki M.A. Trends Cell Biol. 2007; 17: 87-92Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Because Cdc37 partially inhibits the ATPase of Hsp90 (31Siligardi G. Panaretou B. Meyer P. Singh S. Woolfson D.N. Piper P.W. Pearl L.H. Prodromou C. J. Biol. Chem. 2002; 277: 20151-20159Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), it is reasonable to speculate that a transient stalling of the ATPase facilitates substrate transfer onto Hsp90 in general. Up to now, more than a dozen distinct Hsp90 cofactors have been identified (9Richter K. Meinlschmidt B. Buchner J. Buchner J. Kiefhaber T Protein Folding Handbook. Wiley-VCH Verlag GmbH & Co., Weinheim, Germany2005: 768-829Crossref Scopus (1) Google Scholar). Their large number is not paralleled by other chaperone systems. Most bind Hsp90 with submicromolar affinities (Table 1). The major class of these is the TPR domain-containing cofactors, which include the proteins Hop/Sti1, PP5/Ppt1, and the large PPIases, among others (Table 1). Some of these cofactors could specifically facilitate the activation of a certain set of substrate proteins. In this context, the cofactor Unc45 has been shown to participate in early muscle development during the assembly of myosin filaments (32Barral J.M. Hutagalung A.H. Brinker A. Hartl F.U. Epstein H.F. Science. 2002; 295: 669-671Crossref PubMed Scopus (197) Google Scholar), and Xap2/AIP has been found in complex with the protein HbX from the hepatitis B virus (33Kuzhandaivelu N. Cong Y.S. Inouye C. Yang W.M. Seto E. Nucleic Acids Res. 1996; 24: 4741-4750Crossref PubMed Scopus (94) Google Scholar) or the endogenous aryl hydrocarbon receptor (34Meyer B.K. Perdew G.H. Biochemistry. 1999; 38: 8907-8917Crossref PubMed Scopus (175) Google Scholar). It remains to be seen whether these cofactors are really highly specialized or whether they are just the first substrates identified. Interestingly, in yeast, only two cofactors of the Hsp90 system are essential for viability (in addition to Hsp90). These are Cdc37 and Cns1 (29Kimura Y. Rutherford S.L. Miyata Y. Yahara I. Freeman B.C. Yue L. Morimoto R.I. Lindquist S. Genes Dev. 1997; 11: 1775-1785Crossref PubMed Scopus (175) Google Scholar, 35Dolinski K.J. Cardenas M.E. Heitman J. Mol. Cell. Biol. 1998; 18: 7344-7352Crossref PubMed Google Scholar, 36Marsh J.A. Kalton H.M. Gaber R.F. Mol. Cell. Biol. 1998; 18: 7353-7359Crossref PubMed Google Scholar). Cns1 has been shown to associate with both Hsp90 and Hsp70; however, due to the presence of a single TPR domain, no ternary complexes can be formed (37Hainzl O. Wegele H. Richter K. Buchner J. J. Biol. Chem. 2004; 279: 23267-23273Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The function of Cns1 remains elusive. 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The SNF1/AMP-activated protein kinases are central energy regulators in eukaryotes. SNF1 of Saccharomyces cerevisiae is inhibited during growth on high levels of glucose and is activated in response to glucose depletion and other stresses. Activation entails phosphorylation of Thr(210) on the activation loop of the catalytic subunit Snf1 by Snf1-activating kinases. We have used mutational analysis to identify Snf1 residues that are important for regulation. Alteration of Tyr(106) in the αC helix or Leu(198) adjacent to the Asp-Phe-Gly motif on the activation loop relieved glucose inhibition of phosphorylation, resulting in phosphorylation of Thr(210) during growth on high levels of glucose. Substitution of Arg for Gly(53), at the N terminus of the kinase domain, increased activation on both high and low glucose. Alteration of the ubiquitin-associated domain revealed a modest autoinhibitory effect. Previous studies identified alterations of the Gal83 (β) and Snf4 (γ) subunits that relieve glucose inhibition, and we have here identified a distinct set of Gal83 residues that are required. Together, these results indicate that alterations at dispersed sites within each subunit of SNF1 cause phosphorylation of the kinase during growth on high levels of glucose. These findings suggest that the conformation of the SNF1 complex is crucial to maintenance of the inactive state during growth on high glucose and that the default state for SNF1 is one in which Thr(210) is phosphorylated and the kinase is active.