Identification of Amino Acid Residues Required for Ras p21 Target ...

5 downloads 250 Views 2MB Size Report
was kindly provided by I. B. Weinstein, Columbia Univer- sity, New York, N.Y. ...... Housey, G. M., M. D. Johnson, W.-L. W. Hsiao, C. A. O'Brian,. J. P. Murphy, P.
MOLECULAR AND CELLULAR BIOLOGY, Aug. 1991, p. 3997-4004

Vol. 11, No. 8

0270-7306/91/083997-08$02.00/0 Copyright © 1991, American Society for Microbiology

Identification of Amino Acid Residues Required for Ras p21 Target Activation MARK S. MARSHALL,t* LENORA J. DAVIS, ROBERT D. KEYS, SCOTT D. MOSSER, WENDY S. HILL, EDWARD M. SCOLNICK, AND JACKSON B. GIBBS Department of Cancer Research, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486 Received 15 February 1991/Accepted 16 May 1991

The Krev-1 gene has been shown to suppress ras-mediated transformation in vitro. Both ras and Krev-1 proteins have identical effector domains (ras residues 32 to 40), which are required for biological activity and for the interaction of Ras p21 with Ras GTPase-activating protein (GAP). In this study, five amino acid residues flanking the ras effector domain, which are not conserved with the Krev-1 protein, were shown to be required for normal protein-protein interactions and biological activity. The substitution of Krev-1 p21 residues 26, 27, 30, 31, and 45 with the corresponding amino acid residues from Ras p21 resulted in a Krev-1 protein which had ras function in both mammalian and yeast biological assays. Replacement of these residues in Ras p21 with the corresponding Krev-1 p21 amino acids resulted in ras proteins which were impaired biologically or reduced in their affinity for in vitro GAP binding. Evaluation of these mutant ras proteins have implications for Ras p21-GAP interactions in vivo.

The most common of the genetic lesions identified in human cancers are the ras oncogenes. The normal ras genes encode homologous 21-kDa proteins found in association with the plasma membrane (for a review, see reference 2). These proteins share structural and biochemical similarities with the 40-kDa family of G proteins such as specific binding of GDP and GTP and intrinsic GTP hydrolytic activity. Oncogenic variants of the ras proteins invariably contain mutations which increase the GTP-bound form of the protein, suggesting that the Ras-GTP complex is the active form of the protein. Analysis of the bound nucleotide state of ras proteins in vivo supports the hypothesis that the GTP complex of Ras p21 is the active conformation (10, 17, 38). In Saccharomyces cerevisiae, the RAS proteins have been shown to function as regulating components of the cyclic AMP signal transduction cascade (for a review, see reference 16). The ability of S. cerevisiae RAS protein to stimulate adenylyl cyclase is dependent on the binding of GTP (4, 8, 14). Regulation of the bound nucleotide state of RAS protein in S. cerevisiae tightly controls regulation of the cell cycle by adenylyl cyclase. Despite the clear understanding of RAS function in S. cerevisiae, the exact role of ras proteins in mammalian growth control remains uncertain. Detailed analysis of the H-ras protein has been obtained through site-directed mutagenesis and solution of the crystal structure (2, 9, 13, 30, 36, 40, 41, 45). The H-ras protein has distinct functional domains involved in guanine nucleotide binding, magnesium ion coordination, and GTP hydrolysis. Residues 32 to 40 have been shown to be essential for the biological activity of the ras proteins as well as for GTPdependent interaction with the Ras GTPase-activating protein (GAP) (1, 6, 39, 41). Ras GAP has been postulated to be the regulated target of the ras proteins. The ras 32-40 domain has been termed the effector domain and changes conformation when Ras is bound to GTP (30, 36). Recently a cDNA clone capable of suppressing the trans-

formed phenotype of Ki-ras in NIH 3T3 cells was identified. This suppressor gene encodes a Ras-like, 21-kDa guanine nucleotide-binding protein (28) named Krev-1 (also called RaplA or smg p21A; 26, 37). The Krev-1 and ras proteins share extensive homology throughout the guanine nucleotide-binding domains and are identical in the effector domain (residues 32 to 44). Although the ras and Krev-1 proteins have identical effector domains, only the ras protein can cause cellular transformation. It has been proposed that the Krev-1 protein suppresses ras-mediated transformation by competing for binding to the Ras p21 target. Consistent with this proposal is the observation that Krev-1 p21 can bind to the Ras GAP with higher affinity than can Ras p21 (15, 21). Ras residues 32 to 40, while mediating protein binding, are insufficient in the context of the Krev-1 protein to activate the ras signal transduction pathway in vivo. Using rasl Krev-1 chimeras, Zhang et al. demonstrated that transformation of NIH 3T3 cells by Ras p21 was determined by amino acids 21 to 60 and that amino acids 18 to 40 of Krev-1 p21 determined suppression of ras-mediated transformation (47). Because of the similarities between the first 60 amino acids of the two proteins, the divergence responsible for the differences in biological activity must reside within regions 21 to 31 and 45 to 54. Within these stretches of residues are 14 nonconserved amino acids. A distinct region of the ras protein involved in protein-protein interaction outside of the 32-40 domain has been suggested by peptide competition of the Ras p21-GAP interaction (39). A peptide consisting of Ras p21 residues 17 to 32 effectively competed with ras protein for binding to GAP. Significant divergence is found between the 18-32 regions of Ras p21 and Krev-1 p21, again suggesting that the functional divergences between these two proteins might be encoded by this region. In this study, we have attempted to identify specific amino acid residues distinct from amino acid residues 32 to 40 which allow the ras protein to activate a downstream effector pathway. We have analyzed the structures of the regions flanking the 32-40 domain of the H-ras and Krev-1 proteins and identified five distinct amino acid positions evolutionarily conserved among the transforming ras pro-

* Corresponding author. t Present address: Hematology/Oncology Section, Department of Medicine, Indiana University, Indianapolis, IN 46202-5121.

3997

3998

MOL. CELL. BIOL.

MARSHALL ET AL. ZFFZCTOR DOMAIN

Ras

p21

Krev-1 p21

n hi QV VB Y D P T I E D S Y R K Q V i D g e t C 1 L d I i Q Tv Q f V Q ± V* kY D P T I ED S YR K Q V Qv D C q q Cm L e I

T

20

30

40

50

FIG. 1. Comparison of the Ras p21 and Krev-1 p21 effector domains and flanking residues. Conserved residues are in uppercase letters; nonconserved residues are in lowercase letters. Residues felt to be important in target activation are shaded. The putative effector domains are indicated by the black bar.

teins but divergent from the Krev-1 protein. The substitution of these H-ras residues into Krev-1 was sufficient to promote biological activation of the ras pathway in both mammalian cells and S. cerevisiae by the Krev-1 protein. Replacement of these amino acid positions in the ras protein with the corresponding Krev-1 sequence resulted in significant alteration of both the biochemical and biological properties of Ras p21. These five residues, or a subset of them, appear to play a crucial role in activating ras target proteins. MATERIALS AND METHODS

Recombinant DNA methods. Standard recombinant DNA manipulations were used throughout this study (31). The Krev-1 cDNA was a generous gift from M. Noda, Tsukuba Life Science Center, Tsukuba, Japan (29). Plasmid pMV7 was kindly provided by I. B. Weinstein, Columbia University, New York, N.Y. (22). Mutagenesis of H-ras and Krev-1. Mutations were introduced into the H-ras gene by oligonucleotide-directed mutagenesis as previously described (40). Mutant H-ras genes were subcloned into pUC8 for expression in Escherichia coli as lacZ fusion proteins (19, 33) and into the EcoRI site of pMV7 for mammalian expression. Site-directed mutations were introduced into the Krev-1 cDNA by using the Amersham mutagenesis kit. Mutated Krev-1 genes were subcloned into the EcoRI site of pMV7 or the Hindlll site of pAAH5 (42) for mammalian and yeast expression, respectively. In the mutant designations that follow, within brackets are given the amino acid (single-letter code) and position (superscript number) mutated. The [V"2]H-ras"': :Krev6l-183 fusion was made by introducing a silent PstI site at codons 58 and 59 of [V12]H-ras, which allowed recombination with the Krev-1 gene at the corresponding PstI site. The Ki-ras CAAX box coding sequence (CVIM) was added to the [V12]Krev-1 and [V12N26H27D30E31V45]Krev-1 genes, using the polymerase chain reaction to modify the ends of the gene (23). All mutations were confirmed by DNA sequencing using Sequenase enzyme (United States Biochemical). Purification and biochemical analysis of mutant H-ras proteins. Fusion Ras p21 proteins were expressed in E. coli RR1 laCIq and purified as previously described (19, 40). The individual ras proteins were analyzed for intrinsic GTPase activity and GAP-binding affinity as previously described (12, 18, 19, 43). The sensitivity of intrinsic GTPase to GAP was measured as described but using 10 fmol of E. coliproduced bovine GAP in each reaction (18, 33). Mammalian cell culture and transfections. NIH 3T3 cells shown to be Ras p21 dependent for growth were obtained from D. Stacey, Cleveland Clinic, Cleveland, Ohio. Cells were maintained in high-glucose Dulbecco modified Eagle medium (Cellgro) at 37°C in a humidified incubator having 5% CO2. Media was supplemented with 10% calf serum (GIBCO). Cells were transfected with 20 p,g of DNA by

using a calcium phosphate transfection kit (GIBCO BRL), followed on day 3 by splitting 1/10 of the culture into medium supplemented with 1 mg of G418 (geneticin; GIBCO) per ml and the remainder into G418-free medium for focus formation. Foci were subcloned by using cloning cylinders and then enriched in G418-supplemented medium. [3H]thymidine uptake. Cells were seeded at 4 x 104 in medium containing 10% dialyzed calf serum (GIBCO) in 24-well Costar trays. After 24 h, the monolayers were washed with phosphate-buffered saline and medium containing 0.05% dialyzed calf serum was added. After 2 days, 2 ,uCi of 3H-thymidine (1 mCi/ml; Amersham) was added to each well. Cells were incubated for an additional 4 to 6 h before harvesting by trypsinization and filtration through glass fiber filtermats (Skatron). The filters were dried in a microwave oven (1.5 min at medium-high) and counted in scintillation fluid. Duplicate wells were harvested for direct determination of cell count. Radioactivity was expressed as counts per minute per cell number. Biological determination of RAS function in S. cerevisiae. S. cerevisiae 112.699 (ras2 leu2) was transfected to leucine prototrophy with plasmids that constitutively expressed H-ras or mutant Krev-1 genes (32, 42). The ability of each gene to provide RAS function in vivo was determined by scoring the transfected strain for growth on YP-glycerol plates at 37.5°C (7, 32). RESULTS Activation of the transforming potential of Krev-l. The amino-terminal sequences of the ras and Krev-1 proteins were compared, and 14 dissimilar residues were identified in the regions of predicted target activation defined by the peptide competition and raslKrev-1 chimera studies (Fig. 1). The first 19 amino acids of the H-ras and Krev-1 proteins are conserved and were disregarded. Amino acid residues 21, 23, and 24 are within the protein's interior and apparently would not be available for direct protein-protein interaction (9, 36). Dissimilarities were observed between Ras p21 and Krev-1 p21 in the region from amino acids 26 to 31 and 45 to 54. In the 26-31 region, Ras p21 residues N-26, H-27, D-30, and E-31 are located on the surface of the protein and differ in structure or charge from the corresponding amino acids in the Krev-1 protein. The sequence differences observed between residues 45 and 54 are either conservative or at positions which are variable among the ras family of proteins with the exception of V-45. On the basis of this comparison, it seemed possible that Ras residues N-26, H-27, D-30, E-31, and V-45 might constitute an activation domain. To examine the possibility that these five amino acid residues are involved in the activation of the ras pathway, we attempted to convert Krev-1 into a ras-like transforming gene. The Krev-1 gene was mutated to encode H-ras residues at positions 26, 27, 30, 31, and 45. A [V12]Krev-1 gene

VOL. 11, 1991

AMINO ACIDS REQUIRED FOR RAS p21 TARGET ACTIVATION

TABLE 1. Transformation of NIH 3T3 cells by mutant Krev-1 genes Focus formationa Transfection DNA Vector ......................................

3999

A