Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src frequently occurs in human tumorsand is linked to enhanced tumor growth. In experimental systems this synergistic growth requires EGF-dependent association of c-Src with the EGFR and phosphorylation of Tyr-845 of the receptor by c-Src. A search for signaling mediators of Tyr(P)-845 revealed that mitochondrial cytochrome c oxidase subunit II (CoxII) binds EGFR in a Tyr(P)-845-and EGF-dependent manner. In cells this association involves translocation of EGFR to the mitochondria, but regulation of this process is ill-defined. The current study demonstrates that c-Src translocates to the mitochondria with similar kinetics as EGFR and that the catalytic activity of EGFR and c-Src as well as endocytosis and a mitochondrial localization signal are required for these events. CoxII can be phosphorylated by EGFR and c-Src, and EGF stimulation reduces Cox activity and cellular ATP, an event that is dependent in large part on EGFR localized to the mitochondria. These findings suggest EGFR plays a novel role in modulating mitochondrial function via its association with, and modification of CoxII. The epidermal growth factor receptor (EGFR)2 is overexpressed in many cancers including breast cancers, where its overexpression is associated with a poor prognosis (1, 2), yet EGFR alone when overexpressed in fibroblasts is a weak oncogene (3). The non-receptor-tyrosine kinase, c-Src, is also overexpressed in ϳ70% of breast cancers (4), suggesting that EGFR and c-Src may function cooperatively in cancers that co-overexpress both kinases (1). Investigations in murine fibroblasts and in human breast cancer cells have revealed synergistic increases in DNA synthesis, soft agar colony growth, and tumor formation in nude mice when EGFR and c-Src are co-overexpressed as compared with cells overexpressing EGFR or c-Src alone (5, 6). These synergistic increases are dependent upon EGF stimulation, the kinase activity of c-Src, and c-Src phosphorylation of tyrosine 845 (Tyr-845) (7), a highly conserved residue in the activation loop of the catalytic domain of the EGFR. Substitution of a phenylalanine for a tyrosine at position 845 (Y845F) ablates EGF-induced DNA synthesis (even in the presence of overexpressed c-Src) without affecting the intrinsic kinase activity of the EGFR or its ability to phosphorylate canonical downstream substrates (13). Combined, these results suggest that phosphorylated Tyr-845 (Tyr(P)-845) elicits a critical mitogenic signal when EGFR and c-Src are co-overexpressed that is mediated through unconventional substrates.Subsequent efforts to identify mediators of the Tyr(P)-845 EGFR mitogenic signal revealed that phosphorylation and transcriptional activation of Stat5b are dependent on Tyr(P)-845 and that Stat5b is required for EGF-induced DNA synthesis (8). Phage display screening of a human breast cancer tissue library also identified cytochrome c oxidase subunit II (CoxII) as a protein that binds the EGFR in a Tyr(P)-845-and EGF-dependent...
Saccharomyces cerevisiae CDC7 encodes a serine/threonine kinase required for G 1 /S transition, and its related kinases are present in fission yeast as well as in higher eukaryotes, including humans. Kinase activity of Cdc7 protein depends on the regulatory subunit, Dbf4, which also interacts with replication origins. We have identified him1؉ from two-hybrid screening with Hsk1, a fission yeast homologue of Cdc7 kinase, and showed that it encodes a regulatory subunit of Hsk1. Him1, identical to Dfp1, previously identified as an associated molecule of Hsk1, binds to Hsk1 and stimulates its kinase activity, which phosphorylates both catalytic and regulatory subunits as well as recombinant MCM2 protein in vitro. him1؉ is essential for DNA replication in fission yeast cells, and its transcription is cell cycle regulated, increasing at middle M to late G 1 . The protein level is low at START in G 1 , increases at the G 1 /S boundary, and is maintained at a high level throughout S phase. Him1 protein is hyperphosphorylated at G 1 /S through S during the cell cycle as well as in response to early S-phase arrest induced by nucleotide deprivation. Deletion of one of the motifs conserved in regulatory subunits for Cdc7-related kinases as well as alanine substitution of three serine and threonine residues present in the same motif resulted in a defect in checkpoint regulation normally induced by hydroxyurea treatment. The alanine mutant also showed growth retardation after UV irradiation and the addition of methylmethane sulfonate. In keeping with this result, a database search indicates that him1 ؉ is identical to rad35 ؉ . Our results reveal a novel function of the Cdc7/Dbf4-related kinase complex in S-phase checkpoint control as well as in growth recovery from DNA damage in addition to its predicted essential function in S-phase initiation.
Degenerate oligonucleotide‐directed polymerase chain reaction was conducted to clone a possible Schizosaccharomyces pombe homologue [hsk1 for a putative homologue of CDC7 (seven) kinase 1] of Saccharomyces cerevisiae Cdc7 kinase. The cloned cDNA for hsk1+ contains an open reading frame consisting of 507 amino acids with predicted mol. wt of 58,370 that possesses overall amino acid identity of 46% (65% including similar residues) to CDC7. In addition to conserved domains for serine‐threonine kinases, the predicted primary structure of Hsk1 contains three ‘kinase insert’ sequences characteristic to Cdc7 at the positions identical to those of Cdc7. Whereas the length and sequences of the kinase inserts are diverged between the two yeast species, 58% identity (76% including similar residues) is detected within the kinase conserved domains. The hsk1+ gene, which is present as a single copy on the S.pombe chromosome, contains two introns within the coding frame. Disruption of the hsk1+ gene by insertion of the ura4+ gene is lethal to growth. Analysis of the DNA content of germinating spores that contain hsk1 null alleles indicates that DNA replication is inhibited in the mutant. The morphology of these mutant spores after germination indicates abnormal nuclear division in some population of germinating spores, suggesting either that Hsk1 may be required for inhibition of mitosis until completion of S phase or that it may also be involved in proper execution of mitosis. Our results suggest that hsk1+ is a strong candidate for the functional fission yeast homologue of budding yeast CDC7 and that a mechanism through which initiation of chromosomal replication is regulated may be conserved between the two yeast species.
TFIID is a multiprotein complex composed of TBP and several TAF II s. Small amino-terminal segments (TAF Nterminal domain (TAND)) ofYeast strains containing mutant yTAF II 145 lacking yTANDI or yTANDII showed a temperature-sensitive growth phenotype. The conserved core of dTANDII could substitute for the yTANDII core, and Phe-57 or Tyr-129 described above was critically required for the function of this segment in promoting normal cell growth at 37°C. In these respects, the impact of yTAN-DII mutations on cell growth paralleled their effects on TBP binding in vitro, strongly suggesting that the yTAF II 145-TBP interaction and its negative effects on TFIID binding to core promoters are physiologically important.Transcription of protein coding genes in eukaryotes is carried out by RNA polymerase II and a set of auxiliary initiation factors (1, 2). These factors, including TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, can be assembled in a combinatorial fashion in vitro to form a preinitiation complex. Recently, it was proposed that most of these factors are preassembled in vivo in the form of holoenzyme and recruited as a single complex to the core promoter to initiate transcription (3, 4). Barberis et al. (5) reported that recruitment of holoenzyme via a fortuitous interaction between GAL4 DNA binding domain and GAL11 or by fusing lexA to GAL11 would suffice for gene activation in Saccharomyces cerevisiae. A similar result was obtained for SRB2, another component of holoenzyme (6). On the other hand, there is evidence that TBP binding to the TATA box is also a rate-limiting step for transcriptional activation that can be accelerated by gene-specific activators (7). In fact, a physical connection of TBP to a DNA binding module bypasses the requirement for activators (8 -10). Given that TBP is a subunit of TFIID and not a component of holoenzyme (11), it appears that recruitment of either TFIID or holoenzyme will suffice for gene activation in yeast (12). However, it is notable that TFIID is required for both cases because mutation of the TATA sequence greatly decreased activation even by holoenzyme recruitment (5).In higher eukaryotes, the question of how activators stimulate transcription has been addressed mostly by biochemical approaches. Particular attention has focused on TFIID, a multiprotein complex composed of TBP and a series of TBP-associated factors (TAF II s), because TAF II s were shown to be indispensable for activated transcription in vitro (13,14). We and others cloned cDNAs encoding TAF II s from various organisms to decipher the molecular basis of transcriptional regulation (15). It is currently known that TAF II s possess some intriguing structural motifs and/or enzymatic activities. For instance, dTAF II 62/dTAF II 42 forms a histone octamer-like heterotetrameric structure (16). dTAF II 230 has multiple enzymatic activities, including a protein serine kinase activity that selectively phosphorylates RAP74 (17) and a histone acetyltransferase activity specific for histones H3 and H4 (18). Furthe...
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