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P Polakis

Publications and source records attributed to P Polakis.

At least 37 records · Page 2Linked to original sources
PubMed1997

The adenomatous polyposis coli (APC) tumor suppressor.

Defects in the APC gene are inarguably linked to the progression of colon cancers that arise both sporadically and through the transmission of germline mutations. Genetic evidence from humans and mouse models suggest that APC is a classic tumor suppressor in that both alleles likely require inactivation for tumor growth to ensue. Nearly all of the mutations, germline and somatic, result in premature termination of the single polypeptide chain, normally consisting of 2843 amino acids. Several definable motifs have now been mapped to the linear amino acid sequence of the APC polypeptide. These include an oligomerization domain, armadillo repeats, binding sites for beta-catenin, the human discs large protein, microtubules, and other proteins of unknown function. Inactivation of APC in cancer is likely due to loss of function(s) normally associated with the deleted protein structure.

Adenomatous Polyposis Coli↗
PubMed1997

Stabilization of beta-catenin by genetic defects in melanoma cell lines.

Signal transduction by beta-catenin involves its posttranslational stabilization and downstream coupling to the Lef and Tcf transcription factors. Abnormally high amounts of beta-catenin were detected in 7 of 26 human melanoma cell lines. Unusual messenger RNA splicing and missense mutations in the beta-catenin gene (CTNNB1) that result in stabilization of the protein were identified in six of the lines, and the adenomatous polyposis coli tumor suppressor protein (APC) was altered or missing in two others. In the APC-deficient cells, ectopic expression of wild-type APC eliminated the excess beta-catenin. Cells with stabilized beta-catenin contained a constitutive beta-catenin-Lef-1 complex. Thus, genetic defects that result in up-regulation of beta-catenin may play a role in melanoma progression.

Adenomatous Polyposis Coli Protein↗
PubMed1997

Adenomatous polyposis coli tumor suppressor protein has signaling activity in Xenopus laevis embryos resulting in the induction of an ectopic dorsoanterior axis.

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are linked to both familial and sporadic human colon cancer. So far, a clear biological function for the APC gene product has not been determined. We assayed the activity of APC in the early Xenopus embryo, which has been established as a good model for the analysis of the signaling activity of the APC-associated protein beta-catenin. When expressed in the future ventral side of a four-cell embryo, full-length APC induced a secondary dorsoanterior axis and the induction of the homeobox gene Siamois. This is similar to the phenotype previously observed for ectopic beta-catenin expression. In fact, axis induction by APC required the availability of cytosolic beta-catenin. These results indicate that APC has signaling activity in the early Xenopus embryo. Signaling activity resides in the central domain of the protein, a part of the molecule that is missing in most of the truncating APC mutations in colon cancer. Signaling by APC in Xenopus embryos is not accompanied by detectable changes in expression levels of beta-catenin, indicating that it has direct positive signaling activity in addition to its role in beta-catenin turnover. From these results we propose a model in which APC acts as part of the Wnt/beta-catenin signaling pathway, either upstream of, or in conjunction with, beta-catenin.

Adenomatous Polyposis Coli Protein↗
PubMed1997

A Drosophila homolog of the tumor suppressor gene adenomatous polyposis coli down-regulates beta-catenin but its zygotic expression is not essential for the regulation of Armadillo.

Mutations in the adenomatous polyposis coli gene (which encodes a protein called APC) are associated with the formation of intestinal polyps and colon cancers. To facilitate the functional study of APC we have isolated its Drosophila homolog (D-APC) by screening an expression library with an antibody against human APC. The isolated cDNA encodes a predicted 2416-amino acid protein containing significant homology to multiple domains of mammalian APCs. D-APC has seven complete armadillo repeats with 60% identity to its human homolog, one beta-catenin binding site, and up to 7 copies of a 20-amino acid repeat with the average of 50% identity to human APC at amino acid level. D-APC, like its human counterpart, also contains a basic domain. Expression of the domain of D-APC homologous to the region required for beta-catenin down-regulation resulted in down-regulation of intracellular beta-catenin in a mammalian cell line. This same region bound to the Armadillo (Arm) protein, in vitro, the Drosophila homolog of beta-catenin. D-APC RNA and protein expression is very low, if detectable at all, during stages when Arm protein accumulates in a striped pattern in the epidermis of the Drosophila embryos. Removing zygotic D-APC expression did not alter Arm protein distribution, and the final cuticle pattern was not affected significantly. As observed in the rodent, high levels of D-APC expression have been detected in the central nervous system, suggesting a role for D-APC in central nervous system formation.

Adenomatous Polyposis Coli Protein↗
PubMed1997

Induction of phosphorylation on BRCA1 during the cell cycle and after DNA damage.

BRCA1, the familial breast cancer susceptibility gene product, is a 220-kDA phosphorylated protein. BRCA1 immunoprecipitated from MCF7 cells blocked in G1-S phase or progressing through S-phase of the cell cycle migrated more slowly through SDS polyacrylamide gels than BRCA1 from cells maintained in serum-supplemented media, serum-free media for 24 h, or delayed in G2-M phase by treatment with colchicine. Restoration of BRCA1 to the faster-migrating form, which occurred on release of cells from the G1-S-phase block, was prevented by the phosphatase inhibitor okadaic acid. Phosphatase treatment of immunoprecipitated BRCA1 resulted in the conversion of the slower-migrating form to the faster-migrating form. Although these results suggested that BRCA1 was preferentially hyperphosphorylated near the G1-S-phase boundary of the cell cycle, exposure of cells to DNA-damaging agents including UV light or treatment with hydrogen peroxide (H2O2) also promoted BRCA1 hyperphosphorylation. These same stimuli also eliminated the punctate nuclear staining pattern normally observed for BRCA1 in control cells. These results indicate that BRCA1 undergoes cyclic hyperphosphorylation during the cell cycle; however, this modification, as well as changes in BRCA1 nuclear staining, also occurs in response to DNA damage.

Aphidicolin↗
PubMed1996

Subcellular localization and analysis of apparent 180-kDa and 220-kDa proteins of the breast cancer susceptibility gene, BRCA1.

The breast cancer susceptibility gene BRCA1 encodes an 1863-amino acid protein that acts as a tumor suppressor. The biochemical function of BRCA1 is unknown, and there are conflicting results describing its subcellular location. We have identified a 220-kDa protein, which is reactive with three antibodies raised against the amino- and carboxyl-terminal regions of BRCA1. Immunoflourescence staining with an antibody to the carboxyl terminus of BRCA1 localized the protein to the nucleus of breast, ovarian, and cervical carcinoma-derived cell lines. A similar result was observed by biochemical subcellular fractionation that indicated that the 220-kDa protein was localized primarily to the nucleus of cell lines established from breast carcinomas. In addition to the 220-kDa protein, one antibody, C-20, also recognized a 180-kDa protein in MDA-MB-468 total cell lysates that was not detected by the other two antibodies. Several observations suggest the 180-kDa protein is the epidermal growth factor (EGF) receptor: (i) C-20 reacted avidly with a 180-kDa protein immunoprecipitated by an antibody to the EGF receptor; (ii) an EGF receptor antibody detected a 180-kDa protein immunoprecipitated by C-20; (iii) the affinity purified EGF receptor was both immunoprecipitated and detected on immunoblots by the C-20 antibody but not another BRCA1 antibody; (iv) similar phosphopeptide maps were generated from the EGF receptor and the 180-kDa protein immunoprecipitated by C-20, and this peptide map was distinct from the 220-kDa phosphoprotein; and (v) the C-20 immunizing peptide bears sequence identity to the EGF receptor. These results indicate that BRCA1 is a 220-kDa nuclear protein and that the 180-kDa protein reported previously may be unrelated to BRCA1.

Antibodies↗
PubMed1996

Identification of a novel guanine nucleotide exchange factor for the Rho GTPase.

The Rho GTPase promotes proliferation and cytoskeletal rearrangements in mammalian cells. To understand the regulation of Rho, it is important to characterize guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of GDP and subsequent binding of GTP. Using Rho as an affinity ligand, we have isolated a 115-kDa protein (p115-RhoGEF) that binds specifically to the nucleotide-depleted state. A full-length cDNA encoding p115-RhoGEF was isolated, and its protein product, which exhibited sequence homology to Dbl and Lbc, catalyzed the exchange of GDP for GTP specifically on Rho and not on the Rac, Cdc42, or Ras GTPases. p115-RhoGEF is capable of regulating cell proliferation, as determined by its ability to induce the transformation of NIH 3T3 cells. Northern and Western analysis suggests that p115-RhoGEF is ubiquitously expressed. These results indicate that p115-RhoGEF may be a general regulator of Rho and its associated cellular phenotypes.

3T3 Cells↗
PubMed1996

IQGAP1, a calmodulin-binding protein with a rasGAP-related domain, is a potential effector for cdc42Hs.

Proteins that associate with the GTP-bound forms of the Ras superfamily of proteins are potential effector targets for these molecular switches. A 195 kDa protein was purified from cell lysates by affinity chromatography on immobilized cdc42Hs-GTP and a corresponding cDNA was isolated. Sequence analysis revealed localized identities to calponin, the WW domain, unconventional myosins and to the rasGAP-related domain (GRD) contained in IRA, NF-1, SAR1 and rasGAP. p195 was found to be identical to IQGAP1, a protein previously reported to bind ras. Purified recombinant p195/IQGAP1 bound to and inhibited the GTPase activity of cdc42Hs and rac whereas no interaction with ras was detected. The C-terminal half of IQGAP1 containing the GRD bound to cdc42 and rac in a GRD-dependent fashion, but a smaller fragment containing only the GRD did not. Cdc42 was also co-immunoprecipitated from cell lysates with antibody specific to p195/IQGAP1. Calmodulin also co-immunoprecipitated with p195/IQGAP1 and was found to associate with fragments containing the IQ domain. Expression of a cDNA fragment encoding the GRD inhibited the CDC24/CDC42 pathway in yeast, but no effect on ras was observed. In mammalian cells, both endogenous and ectopically expressed p195/IQGAP1 were localized to lamellipodia and ruffling cell membranes, where co-localization with actin was apparent. These results suggest that IQGAP1 is an effector target for cdc42Hs and may mediate the effects of this GTPase on cell morphology.

Amino Acid Sequence↗
PubMed1996

Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly.

The adenomatous polyposis coli gene (APC) is mutated in most colon cancers. The APC protein binds to the cellular adhesion molecule beta-catenin, which is a mammalian homolog of ARMADILLO, a component of the WINGLESS signaling pathway in Drosophila development. Here it is shown that when beta-catenin is present in excess, APC binds to another component of the WINGLESS pathway, glycogen synthase kinase 3beta (GSK3beta), a mammalian homolog of Drosophila ZESTE WHITE 3. APC was a good substrate for GSK3 beta in vitro, and the phosphorylation sites were mapped to the central region of APC. Binding of beta-catenin to this region was dependent on phosphorylation by GSK3 beta.

Adenomatous Polyposis Coli Protein↗
PubMed1996

The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration.

Mutations in the adenomatous polyposis coli (APC) gene are linked to polyp formation in familial and sporadic colon cancer, but the functions of the protein are not known. We show that APC protein localizes mainly to clusters of puncta near the ends of microtubules that extend into actively migrating regions of epithelial cell membranes. This subcellular distribution of APC protein requires microtubules, but not actin filaments. APC protein-containing membranes are actively involved in cell migration in response to wounding epithelial monolayers, addition of the motorgen hepatocyte growth factor, and during the formation of cell-cell contacts. In the intestine, APC protein levels increase at the crypt/villus boundary, where cell migration is crucial for enterocyte exit from the crypt and where cells accumulate during polyp formation that is linked to mutations in the microtubule-binding domain of APC protein. Together, these data indicate that APC protein has a role in directed cell migration.

Adenomatous Polyposis Coli Protein↗
PubMed1996

Wnt-1 regulates free pools of catenins and stabilizes APC-catenin complexes.

The Wnt-1 proto-oncogene induces the accumulation of beta-catenin and plakoglobin, two related proteins that associate with and functionally modulate the cadherin cell adhesion proteins. Here we have investigated the effects of Wnt-1 expression on the tumor suppressor protein APC, which also associates with catenins. Expression of Wnt-1 in two different cell lines greatly increased the stability of APC-catenin complexes. The steady-state levels of both catenins and APC were elevated by Wnt-1, and the half-lives of both beta-catenin and plakoglobin associated with APC were also markedly increased. The stabilization of catenins by Wnt-1 was primarily the result of a selective increase in the amount of uncomplexed, monomeric beta-catenin and plakoglobin, detected both by affinity precipitation and size-exclusion chromatography of cell extracts. Exogenous expression of beta-catenin was possible in cells already responding to Wnt-1 but not in the parental cells, suggesting that Wnt-1 inhibits an essential regulatory mechanism for beta-catenin turnover. APC has the capacity to oppose this Wnt-1 effect in experiments in which overexpression of the central region of APC significantly reduced the size of the monomeric pool of beta-catenin induced by Wnt-1. Thus, the Wnt-1 signal transduction pathway leads to the accumulation of monomeric catenins and stabilization of catenin complex formation with both APC and cadherins.

Adenomatous Polyposis Coli Protein↗
PubMed1996

Deletion of an amino-terminal sequence beta-catenin in vivo and promotes hyperphosporylation of the adenomatous polyposis coli tumor suppressor protein.

Regulation of cell adhesion and cell signaling by beta-catenin occurs through a mechanism likely involving the targeted degradation of the protein. Deletional analysis was used to generate a beta-catenin refractory to rapid turnover and to examine its effects on complexes containing either cadherin or the adenomatous polyposis coli (APC) protein. The results show that amino-terminal deletion of beta-catenin results in a protein with increased stability that acts in a dominant fashion with respect to wild-type beta-catenin. Constitutive expression in AtT20 cells of a beta-catenin lacking 89 N-terminal amino acids (deltaN89beta-catenin) resulted in severely reduced levels of the more labile wild-type beta-catenin. The mutant beta-catenin was expressed at endogenous levels but displaced the vast majority of wild-type beta-catenin associated with N-cadherin. The deltaN89beta-catenin accumulated on the APC protein to a level 10-fold over that of wild-type beta-catenin and recruited a kinase into the APC complex. The kinase was highly active toward APC in vitro and promoted a sodium dodecyl sulfate gel band shift that was also evident for endogenous APC from cells expressing the mutant beta-catenin. Unlike wild-type beta-catenin, which partitions solely as part of a high-molecular-weight complex, the deltaN89 mutant protein also fractionated as a stable monomer, indicating that it had escaped the requirement to associate with other proteins. That similar N-terminal mutants of beta-catenin have been implicated in cellular transformation suggests that their abnormal association with APC may, in part, be responsible for this phenotype.

Adenomatous Polyposis Coli Protein↗
PubMed1995

Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein.

The APC tumor-suppressor protein associates with beta-catenin, a cell adhesion protein that is upregulated by the WNT1 oncogene. We examined the effects of exogenous APC expression on the distribution and amount of beta-catenin in a colorectal cancer cell containing only mutant APC. Expression of wild-type APC caused a pronounced reduction in total beta-catenin levels by eliminating an excessive supply of cytoplasmic beta-catenin indigenous to the SW480 colorectal cancer cell line. This reduction was due to an enhanced rate of beta-catenin protein degradation. Truncated mutant APC proteins, characteristic of those associated with cancer, lacked this activity. Mutational analysis revealed that the central region of the APC protein, which is typically deleted or severely truncated in tumors, was responsible for the down-regulation of beta-catenin. These results suggest that the tumor-suppressor activity of mutant APC may be compromised due to a defect in its ability to regulate beta-catenin.

Adenomatous Polyposis Coli Protein↗
PubMed1995

The APC protein and E-cadherin form similar but independent complexes with alpha-catenin, beta-catenin, and plakoglobin.

The tumor suppressor APC protein associates with the cadherin-binding proteins alpha- and beta-catenin. To examine the relationship between cadherin, catenins, and APC, we have tested combinatorial protein-protein interactions in vivo, using a yeast two-hybrid system, and in vitro, using purified proteins. beta-Catenin directly binds to APC at high and low affinity sites. alpha-Catenin cannot directly bind APC but associates with it by binding to beta-catenin. Plakoglobin, also known as gamma-catenin, directly binds to both APC and alpha-catenin and also to the APC-beta-catenin complex, but not directly to beta-catenin. beta-Catenin binds to multiple independent regions of APC, some of which include a previously identified consensus motif and others which contain the centrally located 20 amino acid repeat sequences. The APC binding site on beta-catenin may be discontinuous since neither the carboxyl- nor amino-terminal halves of beta-catenin will independently associate with APC, although the amino-terminal half independently binds alpha-catenin. The catenins bind to APC and E-cadherin in a similar fashion, but APC and E-cadherin do not associate with each other either in the presence or absence of catenins. Thus, APC forms distinct heteromeric complexes containing combinations of alpha-catenin, beta-catenin, and plakoglobin which are independent from the cadherin-catenin complexes.

Adenomatous Polyposis Coli Protein↗
PubMed1995

Mutations in the APC gene and their implications for protein structure and function.

An enormous number of germline and somatic mutations have been identified in the APC tumor suppressor gene. Nearly all of these mutations result in premature polypeptide chain termination, but the consequences to APC protein function are unknown. Recent advances, including the identification of an oligomerization domain, the localization of several beta-catenin binding sites, some of which down-regulate beta-catenin in vivo, and the identification of a microtubule-binding domain in the carboxy-terminal region of APC, are beginning to provide some clues.

Adenomatous Polyposis Coli Protein↗
PubMed1994

Phosphorylation of Rap1GAP during the cell cycle.

Rap1GAP (for Rap1 GTPase Activating Protein) is an 89 kD protein that highly stimulates the intrinsic GTPase activity of the small GTP binding protein Rap1. It has been shown that Rap1GAP is phosphorylated in vitro by purified p34cdc2 kinase, which regulates the G2/M transition of the cell cycle. In this work, we have studied the phosphorylation of Rap1GAP during the cell cycle and showed that Rap1GAP is phosphorylated in vivo in interphasic and mitotic Hela cells; the electrophoretic mobility of Rap1GAP from mitotic cells is reduced compared with that from interphasic cells, suggesting that the mitotic form of the protein is hyperphosphorylated. As the cdc2 kinase is specifically active during mitosis, we sought to investigate whether it actually phosphorylates Rap1GAP during this phase of the cell cycle. We show that p34cdc2 co-immunoprecipitated from mitotic Hela cell lysates with an anti human cyclin B1 antibody, but not from interphasic cell lysates, is able to phosphorylate efficiently wild-type Rap1GAP, but not a mutant in which the putative consensus site for phosphorylation by the cdc2 kinase (serine 484) has been altered. Moreover, depletion of p34cdc2 from mitotic extracts abolishes the phosphorylation of Rap1GAP by such lysates. These results therefore strongly suggest that Rap1GAP is indeed a substrate of the cdc2 kinase during mitosis. This phosphorylation does not affect the stimulation of the GTPase activity of Rap1 by Rap1GAP but may play a role in regulating the interaction of Rap1GAP with other proteins involved in the cellular functions regulated by Rap1 and Rap1GAP.

Amino Acid Sequence↗