Molecular cloning of the gene for the human placental GTP-binding protein Gp ( G 25 K ) : Identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC 42

We have isolated cDNA clones from a human placental library that code for a low molecular weight GTPbinding protein originally designated Gp (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human Gp protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins (-50% identical), and the rac proteins ('=70% identical). The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell-division-cycle protein CDC42. The human placental gene, which we designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein. GTP-binding proteins play key roles in a number of biological processes, including the hormonal regulation of adenylyl cyclase and phospholipases, visual transduction, protein synthesis, protein trafficking, secretion, and cell growth. The members of the growing class of low molecular weight GTP-binding proteins, with the ras oncogene products as prototypes, consist of only a single 21to 28-kDa polypeptide (1). These proteins include the yeast YPT1 and SEC4 proteins, which have been implicated in transport through the Golgi and exocytosis (2, 3); the mammalian rho proteins, which are substrates for ADP-ribosylation by botulinum toxin (4, 5); the mammalian rap (6-8), rab (9), ral (10), and rac (10, 11) proteins; the bovine and yeast ARF proteins (12), which promote the cholera toxin-induced ADP-ribosylation of GU in the Golgi compartment; and the human bovine Gp (G25K) proteins (13, 14). The well-known transforming properties of the ras oncogenes (15) and the recent observation that the raplA protein can counteract ras transformation (8) suggest that members of this protein family play key roles in cell growth pathways. Recently the Gp protein was shown to be an excellent substrate for the tyrosine kinase activity of the purified human placental epidermal growth factor (EGF) receptor (16), which suggests the possible involvement of this protein in the actions of the EGF receptor. We have now cloned a full-length cDNA for Gp from a human placental library. Analysis of the amino acid sequence and functional studies of the expressed protein indicate that Gp is the mammalian homolog of a yeast cell-division-cycle protein, CDC42. ¶ MATERIALS AND METHODS Isolation of GP cDNA Clones. A human full-term placental cDNA library in Agtl1 was kindly provided by Evan Sadler (Washington University School of Medicine). Two oligonucleotide probes (P1 and P4; see Results) based on the peptide sequences ofthe purified human platelet and placental Gp (14) were labeled using [y-32P]ATP and T4 kinase. A third oligonucleotide probe (P1') was prepared from a cDNA clone that was isolated from the placental cDNA library (see Results). Hybridization was carried out under low stringency at 420C in 6x SSC (1x is 0.15 M NaCJ/0.015 M sodium citrate, pH 7)/5x Denhardt's solution (1x is 0.02% Ficoll/0.02% polyvinylpyrrolidone/0.02% bovine serum albumin)/1% SDS/50 mM Tris'HCI, pH 7.5, containing denatured salmon sperm DNA at 50 tug/ml. Filters were washed in 2 x SSC at 200C and then in 0.2x SSC/0.1% SDS at 500C. Hybridizing phages were plaque-purified and confirmed positive by Southern hybridization with the various probes. The cDNA inserts were subcloned into plasmid pUC19 and both strands were sequenced by the dideoxy chain-termination procedure (17). Sequence homology alignments were performed by the method of Lipman and Pearson (18). Expression of CDC42Hs in Saccharomyces cerevisiae. The conditions for the growth and maintenance of S. cerevisiae strain DJTD2-16A (MATa cdc42-1 ura3 his4 leu2 trpl) have been described (19). The permissive and restrictive temperatures for the growth of DJTD2-16A were 230C and 36°C, respectively. The plasmid pBM272, which has the URA3 yeast selectable marker and the GALIO promoter (provided by M. Johnston, Univ. of Vermont), was used to express CDC42Hs. The insertion of the coding region for the CDC42Hs cDNA into pBM272 and the results obtained from complementation studies are described in the legend to Table 1. Expression of CDC42Hs in Escherichia coli. The E. coli pGEX plasmids express foreign DNA sequences as glutathione S-transferase (GST) fusion proteins (20). The pGEXCDC42Hs plasmid was constructed by the ligation of a 900base-pair (bp) BamHI-EcoRI fragment of CDC42Hs cDNA into the pGEX-2T plasmid. The BamHI site was created immediately upstream of the CDC42Hs start codon by use of the polymerase chain reaction (PCR) and the EcoRI site was in 3' nontranslated cDNA. The fusion protein was produced in E. coli strain XL1-B and purified as described (21). Abbreviations: EGF, epidermal growth factor; GST, glutathione S-transferase; GTP y-35S], guanosine 5'-[y-[35S]thioltriphosphate; PCR, polymerase chain reaction. tVisiting scientist from Biochemical Research Laboratories, Kanegafuchi Chemical Industry Co. Ltd., Takasago, Hyogo, Japan. The sequence reported in this paper has been deposited in the GenBank data base (accession no. M57298). 9853 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. D ow nl oa de d by g ue st o n A ug us t 4 , 2 02 0 9854 Biochemistry: Shinjo et al. Proc. Natl. Acad. Sci. USA 87 (1990) The CDC42Hs protein was also expressed in E. coli using the pET-3A expression vector under the control of the T7 promoter (22). The PCR was used to create an Nde I site on a 900-bp fragment of the CDC42Hs cDNA and the resulting fragment was cloned into the Nde I/BamHI sites of the pET vector. After induction with isopropyl P3-D-thiogalactopyranoside, the cells were harvested and lysed as described (21). The homogenate was centrifuged at 12,000 x g for 5 min and the supernatant was used for Western immunoblotting experiments. Western blot analyses of the recombinant CDC42Hs protein were performed as described by Polakis et al. (14), and binding of guanosine 5'-[y-[35S]thio]triphosphate (GTP[y-35S]) was measured as outlined by Hart et al. (16).