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Structural Analysis of the Ras Gtpase Activating Protein Catalytic Domain by Semirandom Mutagenesis: Implications for a Mechanism of Interaction with Ras-GTP1

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Structural Analysis of the Ras Gtpase Activating Protein Catalytic Domain by Semirandom Mutagenesis: Implications for a Mechanism of Interaction with Ras-GTP1 [CANCERRESEARCH54. 5438-5444, October 15, 1994] Structural Analysis of the Ras GTPase Activating Protein Catalytic Domain by Semirandom Mutagenesis: Implications for a Mechanism of Interaction with Ras-GTP1 Lisa Hettich and Mark Marshall2 Department of Medicine, Division of Hematology/Oncology. Indiana UniversüySchool of Medicine and Waither Oncology Center, Indianapolis. indiana 46202 ABSTRACT region of GAP in the regulation of cytoskeletal structure and cell adhe sion to the extracellular matrix (14). Expression of the GAP NH2- The bovine complementary DNA encoding the catalytic domain of Ras terminal SH2-SH3-SH2 region in Rat-2 fibroblasts resulted in a disrup GTPase activating protein was mutagenized semirandomly using a van tionofactinstressfibersandfocalcontacts.Thiseffectisprobablycaused atlon of the polymerase chain reaction. Sixty-four mutated codons were identified with seventeen of the mutations deleterious to Ras GTPase by the action of the GAP-associated protein, p190, and Rho (15-17). activating function. All of the inactivating single mutations affected the Changes in the cytoskeleton and loss of adhesion are important compo structure of the catalytic fragment as assessed by large decreases in nents of cell growth. soluble protein when expressed in Escherichia coli. Upon exnmlnation of In addition to its potential as a Ras effector molecule, GAP is the Ran binding properties of 10 of the mutants, only 1 was measurably currently the best model for examining GTP-dependent protein inter Impaired for I(as binding and 4 appeared to have increased affinity for actions with Ras. Ras-GTP has been shown to bind to GAP, while Ran. These results demonstrate that Ras binding and GTPase activation Ras-GDP does not (18). In addition, many mutations within the are two distinct properties of GTPase activating protein. Additionally, the so-called Ras effector region, which block cellular transformation, catalytic mechanism of GTPase activating protein Is much more sensitive also reduces the GAP sensitivity of the GTPase of the mutant or the to structural perturbation than is Ras binding. ability of the protein to bind GAP (19, 20). The primary Ras binding site of GAP has been localized to the carboxy-terminal 343 amino INTRODUCTION acids and is referred to as the GAP catalytic domain (21). This region Mutations in the ras proto-oncogenes have been implicated in the can be expressed as a stable truncated protein in Escherichia coli, initiation and proliferation of tumors in both animal carcinogenesis which can bind Ras-GTP and activate the Ras GTPase. The Ras models and human cancer (1). Oncogenic mutations result in a mod binding domain of GAP is conserved in other eukaryotic proteins ified form of the Ras protein, which is refractory to negative regula possessing Ras-GTPase activating function. It is not known if Ras tion, resulting in a constant proliferative signal (2, 3). Ras is a small binds a smallregionof GAPanalogousto the Ras effectorregionor Mr 21,000 membrane-associated protein which shares many biochem a larger area of the surface typical of most protein-protein interactions. ical characteristics with the a subunits of the prototypical heterotri The goal of this study has been to use a structurally unbiased meric 0-proteins (4). Ras binds both GDP and GTP with high affinity, approach in order to identify specific amino acid residues involved in possesses an intrinsic GTPase, and requires membrane association for the binding of GAP to Ras. Identification of discrete sites of protein normal function. Current evidence suggests that the primary mito interaction on the Ras protein suggests that analogous sites may exist genic effector of Ras is the Raf kinase. Ras-GTP binds to the Raf on GAP. The DNA encoding the catalytic fragment of GAP was serine/threonine kinase and is believed to assist in its activation (5—8). mutagenized using a semirandom method; mutant proteins were iden GAP3 is known to function as a negative regulator of Ras through the tified and biochemically characterized. Inactivating mutations were dramatic stimulation of the slow intrinsic GTPase of the Ras protein. found in 17 distinct codons, while mutations in 47 other codons had In addition to being a negative Ras regulator, GAP has also been little effect. All of the inactivating single codon mutations were found implicated as a component necessary for some Ras-dependent effects in to significantly reduce the quantity of soluble protein produced in mammalian cells and amphibian oocytes. In vitro evidence for a Ras E. coli, suggesting that gross conformational changes had occurred. dependent effector role for GAP has been demonstrated in at least three Only one of these mutants was found to have reduced binding affinity separate model systems. Ras and GAP cooperate in an SH2/5H3 domain for Ras. These results suggest that GAP interacts with Ras, either dependent manner to inhibit muscarinic gated potassium channel activa through a few specific contacts scattered throughout the primary tion in patch-clamped atrial membranes (9, 10). Activation of Xenopus amino acid sequence or through a large surface which is unaffected by 1@zevisp34 kinase (maturation promoting factor) by Ras has also been point substitutions. The activation of Ras GTPase by GAP is much suggested to be GAP-dependent (11), with Ras induction of germinal easier to disrupt than binding alone, indicating that GTPase stimula vesicle breakdown requiring the GAP SH3 domain (12). GAP has also tion requires a more conformationally defined structure which is not been implicated by expression-competition experiments as having posi required for binding. five transcriptional effects on the Ras-dependent polyoma enhancer (13). The most conclusive evidence for a signaling role for GAP comes from MATERIALS AND METHODS a series of experiments that showed involvement of the NH2-terminal Mutagenesis of the GAP gene Semirandom base misincorporations were Received 6/2/94; accepted 8/17/94. introduced into the bovine GAP complementary DNA by PCR using Taq The costs of publication of this article were defrayed in part by the payment of page polymerase (Perkin-Elmer; Ref. 22). The reaction conditions used were as charges. This article must therefore be hereby marked advertisement in accordance with recommended by the manufacturer except that the concentration of dATP was 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported in part by the Project Development Program, Research and limiting (40 @LMversus200 pM dGTP, dTFP, and dCTP). Reduction of dATP Sponsored Programs, Indiana University at Indianapolis. resulted in limited random misincorporation of deoxyribonucleotides when 2 To whom requests for reprints should be addressed, at Walther Oncology Center, ever an “A―(deoxyadenosine)was required during primer extension of the lndiana University School of Medicine, 975 West Walnut Street, Room 501, Indianapolis, DNA. The addition of 0.1 mMMnCl2to the reaction further increased the rate 1N 46202-5121. of base misincorporation. Use of the oligonucleotide primers 5'-GCCCATA 3 The abbreviations used are: GAP, GTPase activating protein; PCR, polymerase chain reaction. AACFCCCAGTAAAG-3' and 5'-CGCO@GCAGAAUAGCfCACACAT 5438 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1994 American Association for Cancer Research. RANDOM MUTAGENESIS OF RASGAP CACI'G-3' amplified only the portion of the bovine GAP gene, which encodes (Qiagen) for 1 h at 4°C.Theagarose was then washed twice in batch with 50 the catalytic domain from codon 701 to 1044. The mixture of mutant GAP ml of buffer A, packed into a small column, and connected to a Pharmacia DNA fragmentswas subclonedfrom the PCR reactioninto the BamHI-PstI FPLC pump and detector. After further washing, bound proteins were eluted sites ofpU@8 in-frame with the lacZ gene to yield an E. coli expression library with a 0—0.250 M imidazole gradient. The A@-absothing peak was concen of GAP[701—1044]mutants.Approximately 80% of the codons in this GAP trated in a Centricon-lO concentrator (Amicon), washed twice with TED, gene fragment had an “A―ora “r'inthe first or second position, making them assayed for GAP activity, and immunoblotted for GAP protein. Although susceptible to amino acid substitution. To enable affinity purification of wild-type GAP[701—1044Jwastypically purified to 90—95%homogeneity,the selected mutant proteins, mutated genes were subcloned as BamHI-PstI frag yield and purity of the mutant proteins were more variable. ments into the pQE-30 expression plasmid (Qiagen). The pQE-30 plasmid Binding of Ran to Mutant GAP PrOteins. The binding of Ras to catalyt provided for high-level expression of proteins fused to the 6x(histidine) affin ically impaired GAP mutants was measured using a kinetic competition assay ity tag. The F898S mutation was separated from a second mutation in the 4-26 (19) which has been previously adapted for comparing the relative Ras binding mutant by subcloning a restriction fragment containing the point change into aff@mityof normal and reduced activity NFl mutants (24—26). Basically, a the wild-type pQE-30-GAP[701-1044] plasmid. Standard recombinant DNA small quantity of [‘y-32P]GTP-boundRas protein (0.2 nM) was incubated in the manipulations were followed throughout this study (23). Plasmid DNAS were presence of sufficient GAP or GAP mutant to stimulate hydrolysis of completely sequenced using Sequenase (United States Biochemical). [y-32P]GTP bound to Ras (approximately 50—200 @g/mlfor most GAPs, Isolation and Preparation of GAP Mutants Single-colony isolates were including the wild-type control; 400—1,200 @g/mlformutants 4-33, 4-150, obtained from the mutated pUC8-GAP[701—1044]expressionlibraries by 4-119, and 4-16). Increasing concentrations of nonradiolabeled Ras[L61]-GTP transformation of RRllaci―E.coli. Individual colonies were transferred into 5 (0.006—200 ELM)were added to the assay mixture described above. All re ml of LBA (Luria broth with 100 mg/ml ampidillin; Ref.
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