Biomedical subjects

P Polakis

Publications and source records attributed to P Polakis.

53 records · Page 3Linked to original sources

PubMed1994

The APC gene product associates with microtubules in vivo and promotes their assembly in vitro.

Defects in the APC gene occur frequently in patients with familial adenomatous polyposis coli and are associated with the progression of sporadic tumors of the colon and stomach. We examined the subcellular location of adenomatous polyposis coli (APC) protein resulting from transient expression of full length and partial APC complementary DNAs in epithelial cells. Immunofluorescent detection revealed an association of APC with cytoplasmic microtubules. Expression of partial complementary DNA constructs indicated that the carboxy-terminal region of the APC protein, typically deleted in cancers, is essential for this association. The same APC polypeptides that associated with microtubules in vivo also dramatically promoted their assembly in vitro. These results suggest that wild-type APC protein binds to and affects the assembly of microtubules, whereas the mutants identified in tumors have lost this activity.

Adenomatous Polyposis Coli Protein↗

PubMed1993

Association of the APC gene product with beta-catenin.

Mutations in the human APC gene are linked to familial adenomatous polyposis and to the progression of sporadic colorectal and gastric tumors. To gain insight into APC function, APC-associated proteins were identified by immunoprecipitation experiments. Antibodies to APC precipitated a 95-kilodalton protein that was purified and identified by sequencing as beta-catenin, a protein that binds to the cell adhesion molecule E-cadherin. An antibody specific to beta-catenin also recognized the 95-kilodalton protein in the immunoprecipitates. These results suggest that APC is involved in cell adhesion.

Adenomatous Polyposis Coli Protein↗

PubMed1993

Structural requirements for the interaction of p21ras with GAP, exchange factors, and its biological effector target.

From the multitude of mutations that have been tested in p21ras, a common theme emerges regarding its interaction with the effector target, GAPs, and the newly discovered exchange factors. Many of the mutations that result in dysfunction for all three types of interactions are localized to the switch 1 and switch 2 regions of the p21ras three-dimensional structure (Fig. 2). These two regions change conformation on GTP binding by p21ras and, accordingly, both GAP binding and Ras biological activity are GTP-dependent processes. In addition, certain mutations in the switch 1 and 2 regions alter the affinity of GAP for p21ras, again implicating this region in the binding interaction. On the other hand, the SDC25 exchange factor appears to promote dissociation of both GTP and GDP from p21ras, suggesting that the overall conformation of the switch 1 and 2 regions may not be important for recognition by SDC25. Moreover, none of the switch 1 or 2 mutations that impaired stimulation by SDC25 affected its binding to p21ras. This suggests that these residues are essential for the mechanism of activation by SDC25 but not for its binding to p21ras. Amino acids at positions 73, 75, and possibly 102 and 103 appear also to be involved in the activation of p21ras by exchange factors. Whether these are required for binding to the exchanger has not been reported.

Animals↗

PubMed1992

Regulation of the GTPase activity of the ras-related rap2 protein.

The small GTP-binding protein rap2A exhibits a high level of identity with rap1 and ras proteins (60% and 46%, respectively). Nevertheless, its intrinsic GTPase activity is not stimulated by ras-GAP, and unlike the rap1A protein, it cannot compete with ras proteins for their interaction with ras-GAP. In addition, rap1-GAPm that is highly active on the GTPase activity of the rap1A product, also stimulates the GTPase activity of the rap2A protein but with a 30-40-fold lower efficiency. An activity that greatly stimulated the GTPase activity of the rap2 protein (rap2-GAP) was found in bovine brain cytosol and purified. However, it copurified with the cytosolic form of rap1-GAP and was more efficient at stimulating the GTPase activity of the rap1 protein; this 55 kD polypeptide, that is recognized by an antibody raised against rap1-GAPm, likely represents a degraded and soluble form of the full size 89 kD molecule. In bovine brain membranes, a weak GAP activity toward the rap2A protein was also detected; however, it was also attributable to the membrane-associated rap1-GAPm. Thus, it appears that a single rap-GAP protein, complete or degraded, is able to stimulate the GTPase activity of both rap1 and rap2 proteins.

Animals↗

PubMed1992

Phosphorylation of rap1GAP in vivo and by cAMP-dependent kinase and the cell cycle p34cdc2 kinase in vitro.

rap1GAP is a GTPase activating protein that specifically stimulates the GTP hydrolytic rate of the ras-related protein p21rap1.rap1GAP undergoes post-translational modification that causes a substantial change in its mobility on sodium dodecyl sulfate-polyacrylamide gels. At least part of this modification is due to the phosphorylation. Expression of a rap1GAP cDNA in insect cells labeled with 32Pi resulted in high level incorporation of radioactivity into serine residues of the expressed protein. Purified rap1GAP was phosphorylated in vitro by cAMP-dependent kinase and the cell cycle p34cdc2 kinase. The molar ratio of incorporated phosphate/rap1GAP was approximately 3 by cAMP-dependent kinase and 2 by p34cdc2. The sites of phosphorylation by both kinases were localized to a 100-residue segment contained in the carboxyl-terminal region of the predicted primary structure of rap1GAP. Highly favorable recognition sequences for the two kinases are contained within this fragment and are proposed as the sites of phosphorylation. Treatment of SK-MEL-3 cells with dibutyryl cAMP promoted phosphorylation of rap1GAP in vivo. Based on the results of comparative phosphopeptide mapping the sites of phosphorylation in vivo and in vitro are identical.

Amino Acid Sequence↗

PubMed1992

Molecular cloning and nucleic acid binding properties of the GAP-associated tyrosine phosphoprotein p62.

p62 is a tyrosine phosphoprotein that associates with p21ras GTPase-activating protein (GAP). Purification and cDNA cloning of p62 reveal extensive sequence similarity to a putative hnRNP protein, GRP33. Recombinant human p62 purified from insect Sf9 cells binds to DNA and to mRNA and, like many proteins involved in mRNA processing, recombinant p62 is modified by dimethylation on multiple arginine residues. p62 also binds tightly to p21ras GAP in vitro: this binding depends on phosphorylation of p62 on tyrosine residues and occurs through SH2 regions of GAP. These data suggest that p120-GAP and p62 play a role in some aspect of mRNA processing or utilization and that this role may be regulated by tyrosine phosphorylation, and indirectly, by p21ras.

3T3 Cells↗

PubMed1992

GAP domains responsible for ras p21-dependent inhibition of muscarinic atrial K+ channel currents.

The interaction between the low molecular weight G protein ras p21 and a guanosine triphosphatase activating protein (GAP) uncouples a heterotrimeric G protein (Gk) from muscarinic receptors. Through the use of isolated atrial cell membranes and genetically engineered GAP deletion mutants, the src homology regions (SH2-SH3) at the amino terminus of GAP have been identified as the domains responsible for this effect. Deletion of the domain required to stimulate the guanosine triphosphatase activity of ras p21 relieves the requirement for ras p21 in this system. A model is presented that suggests that ras p21 induces a conformational change in GAP, which allows the SH2-SH3 regions of GAP to function.

Animals↗

PubMed1992

Localization of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis.

rap1GAP is a GTPase-activating protein that specifically stimulates the GTP hydrolytic rate of p21rap1. We have defined the catalytic domain of rap1GAP by constructing a series of cDNAs coding for mutant proteins progressively deleted at the amino- and carboxy-terminal ends. Analysis of the purified mutant proteins shows that of 663 amino acid residues, only amino acids 75 to 416 are necessary for full GAP activity. Further truncation at the amino terminus resulted in complete loss of catalytic activity, whereas removal of additional carboxy-terminal residues dramatically accelerated the degradation of the protein in vivo. The catalytic domain we have defined excludes the region of rap1GAP which undergoes phosphorylation on serine residues. We have further defined this phosphoacceptor region of rap1GAP by introducing point mutations at specific serine residues and comparing the phosphopeptide maps of the mutant proteins. Two of the sites of phosphorylation by cyclic AMP (cAMP)-dependent kinase were localized to serine residues 490 and 499, and one site of phosphorylation by p34cdc2 was localized to serine 484. In vivo, rap1GAP undergoes phosphorylation at four distinct sites, two of which appear to be identical to the sites phosphorylated by cAMP-dependent kinase in vitro.

Amino Acid Sequence↗

PubMed1992

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae.

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

Amino Acid Sequence↗

PubMed1992

Interactions between p21ras proteins and their GTPase activating proteins.

Two proteins that regulate p21ras GTPase activity have been identified. These proteins interact with a region of ras p21 that is necessary for p21ras function and may themselves be components of signalling complexes. The first of these proteins to be identified, GAP, contains domains that interact with receptor tyrosine kinases and other tyrosine phosphoproteins, providing a direct link between signalling pathways involving these proteins and p21ras. The second, the product of the NF1 gene, is less well characterized but seems to connect p21ras to other signalling pathways which are perturbed in the NF1 disease. The ability of p21ras to interact with GAP may be compromised by competitive binding to the product of the Ki-rev1 gene, p21rap1. This competition for binding to GAP, or other proteins that interact with the effector site of ras p21, may explain the ability of Ki-rev1 to suppress cellular transformation by ras oncogenes.

GTPase-Activating Proteins↗

PubMed1992

Association of a tyrosine kinase activity with GAP complexes in v-src transformed fibroblasts.

p21ras GAP is phosphorylated on tyrosine residues and associates with 62 kDa and 190 kDa tyrosine phosphorylated proteins in v-src-transformed fibroblasts. We were interested in identifying the tyrosine kinase responsible for phosphorylation of GAP and the two associated proteins. Here, we report that GAP-immunoprecipitates from v-src transformed cells contain a tyrosine kinase activity that phosphorylates GAP, p62 and p190. Tryptic peptide analysis indicated that the sites phosphorylated in vitro and in vivo are indistinguishable, suggesting that the precipitated kinase could be responsible for tyrosine phosphorylation of GAP in vivo. The GAP-associated kinase activity might be due to v-src itself, because pp60v-src is able to associate with GAP in vitro and GAP can be phosphorylated by pp60v-src immunecomplexes.

3T3 Cells↗

PubMed1991

Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1.

The rap1/Krev-1 gene encodes a ras-related protein that suppresses transformation by ras oncogenes. We have purified an 88 kd GTPase activating protein (GAP), specific for the rap1/Krev-1 gene product, from bovine brain. Based on partial amino acid sequences obtained from this protein, a 3.3 kb cDNA was isolated from a human brain library. Expression of the cDNA in insect Sf9 cells resulted in high level production of an 85-95 kd rap1GAP that specifically stimulated the GTPase activity of p21rap1. The complete deduced amino acid sequence is not homologous to any known protein sequences, including GAPs specific for p21ras. Northern and Western blotting analysis indicate that rap1GAP is not ubiquitously expressed and appears most abundant in fetal tissues and certain tumor cell lines, particularly the Wilms' kidney tumor, SK-NEP-1, and the melanoma, SK-MEL-3, cell lines.

Amino Acid Sequence↗

PubMed1991

Regulation of ras p21 by GTPase activating proteins.

We propose a model for dual effector functions of the known ras GAPs p120-GAP and NF1-GAP. This model is consistent with known biological and biochemical effects of GAPs in mammalian cells, but it is clearly not a proven hypothesis, and several difficulties remain in making this model convincing. One is the apparent difference between mammalian cells and yeasts, in which GAPs do not have a demonstrable effector function. The other is the difficulty of eliminating the possibility that other effectors exist that do not have GAP activity and do not bind ras p21 sufficiently tightly to allow detection through physical association. We hope that further analysis of GAP function will clarify the roles of these proteins, allowing at least a partial description of ras action in normal and malignant mammalian cells.

Animals↗

PubMed1991

A synthetic peptide corresponding to a sequence in the GTPase activating protein inhibits p21ras stimulation and promotes guanine nucleotide exchange.

Amino acid sequence homology between the GTPase Activating Protein (GAP) and the GTP-binding regulatory protein, Gs alpha, suggests that a specific region of GAP primary structure (residues 891-898) may be involved in its stimulation of p21ras GTP hydrolytic activity (McCormick, F. [1989] Nature 340, 678-679). A peptide, designated p891, corresponding to GAP residues 891-906 (M891RTRVVSGFVFLRLIC906) was synthesized and tested for its ability to inhibit GAP-stimulated p21ras GTPase activity. At a concentration of 25 microM, p891 inhibited GAP activity approximately 50%. Unexpectedly, p891 also stimulated GTP binding to p21N-ras independent of GAP. This stimulation correlated with an enhancement of p21N-ras.GDP dissociation; an approximate 15-fold increase in the presence of 10 microM p891. In contrast, dissociation of the p21N-ras.GTP gamma S complex was unaffected by 10 microM p891. The p21N-ras.GDP complex was unresponsive to 100 microM mastoparan, a peptide toxin shown previously to accelerate GDP dissociation from the guanine nucleotide regulatory proteins, Gi and Go. p21H-ras, as well as the two p21H-ras effector mutants, Ala-38, and Ala-35, Leu-36, also exhibited increased rates of GDP dissociation in the presence of p891. Also tested were three ras-related GTP-binding proteins; rap, G25K and rac. The rap.-GDP complex was unaffected by 10 microM p891. Dissociation of the G25K- and rac.GDP complexes were enhanced slightly; approximately 1.3- and 1.8-fold over control, respectively. Thus, the inhibitory effect of p891 on GAP stimulation of p21ras suggests that amino acids within the region 891-906 of GAP may be essential for interaction with p21ras. In addition, p891 independently affects the nucleotide exchange properties of p21ras.

Amino Acid Sequence↗

PubMed1991

Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activating protein.

The p21ras GTPase-activating protein (GAP) down-regulates p21ras by stimulating its intrinsic GTPase activity. GAP is found predominantly as a monomer in the cytosol of normal cells. However, in cells expressing an activated cytoplasmic protein-tyrosine kinase, p60v-src, or stimulated with epidermal growth factor, GAP becomes phosphorylated on tyrosine and serine and forms distinct complexes with two phosphoproteins of 62 and 190 kDa (p62 and p190). In v-src-transformed Rat-2 cells, a minor fraction of GAP associates with the highly tyrosine phosphorylated p62 to form a complex that is localized at the plasma membrane and in the cytosol. In contrast, the majority of GAP enters a distinct complex with p190 that is exclusively cytosolic and contains predominantly phosphoserine. Epidermal growth factor stimulation also induces a marked conversion of monomeric GAP to higher-molecular-weight species in rat fibroblasts. The GAP-p190 complex is dependent on phosphorylation and shows reduced GAP activity. These results indicate that protein-tyrosine kinases induce GAP to form multiple heteromeric complexes, which are strong candidates for regulators or targets of p21ras.

Animals↗

PubMed1990

ras p21 and GAP inhibit coupling of muscarinic receptors to atrial K+ channels.

The signal-transducing G protein Gk couples muscarinic receptors to K+ (K+[ACh]) channels in atrial cells. Recombinant human ras p21 GAP (GTPase activating protein) at subnanomolar concentrations inhibited GTP-dependent channel opening in isolated atrial cell membranes. This inhibition depended on interaction of GAP with ras p21 in the isolated membranes. In addition, recombinant ras p21 proteins blocked the currents; this effect could be blocked by prior incubation of membranes with specific anti-GAP antibodies. We therefore propose that ras p21 GTP complexed with GAP (ras p21-GAP) blocks K+[ACh] currents. The channel block could be overcome by GTP gamma S activation of endogenous Gk; this indicates that ras p21-GAP does not interfere with interaction of Gk with the K+[ACh] channel directly, but prevents coupling of the muscarinic receptor to endogenous Gk.

Animals↗

PubMed1990

Molecular cloning and expression of a G25K cDNA, the human homolog of the yeast cell cycle gene CDC42.

G25K is a low-molecular-mass GTP-binding protein with a broad distribution in mammalian tissues. A cDNA clone was isolated by using oligonucleotides corresponding to the partial amino acid sequence of purified human G25K. The cDNA encodes an 191-amino-acid polypeptide containing GTP-binding consensus sequences and a putative farnesylation site at the C terminus. The sequence exhibits 50 and 70% identities to the mammalian rho and rac proteins, respectively, and an 80% identity to the Saccharomyces cerevisiae CDC42 gene product. Insect Sf9 cells infected with recombinant baculovirus vectors expressing the G25K cDNA produced a 25-kDa protein that bound GTP and was recognized by antibodies specifically reactive to G25K. G25K appears to be the human homolog of the CDC42 gene product, since expression of the G25K cDNA in S. cerevisiae suppressed both cdc42-1 and cdc24-4 temperature-sensitive lethal mutations.

Amino Acid Sequence↗