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

Cell, Vol. 65, 1033-1042,

June

14, 1991, Copyright

0 1991 by Cell Press

Molecular Cloning of a GTPase Activating Specific for the Krev-I Protein p2VaP7 Bonnee Rubinfeld, Susan Munemitsu, Robin Clark, Leah Conroy, Kenneth Watt, Walter J. Crosier, Frank McCormick, and Paul Polakis Cetus Corporation 1400 53rd Street Emeryville, California 94608

Summary The rapi/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 rapl/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 raplGAP that specifically stimulated the GTPase activity of ~21”~‘. The complete deduced amino acid sequence is not homologous to any known protein sequences, including GAPS specific for ~21”“. Northern and Western blotting analysis indicate that raplGAP 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. Introduction Proteins that bind and hydrolyze GTP are integral components of the cell signaling matrix (reviewed in Bourne et al., 1990; Hall, 1990a). The activities of these molecules are controlled by proteins that regulate their GTP-binding and GTP-hydrolyzing activities. Identification of proteins that specifically regulate the individual members of the ras-related family of GTPases will be necessary to understand the signaling networks in which these molecules operate. One such regulatory protein specific for p21raS, termed GAP (for GTPase activating protein), has been isolated, and cDNAs encoding it have been cloned from human placenta (Trahey et al., 1988) and from bovine brain (Vogel et al., 1988). That GAP has been shown to associate physically with the PDGF receptor (Kaplan et al., 1990; Kazlauskas et al., 1990) is consistent with previous evidence implicating p21”” in transmission of growth factor responses (Hagag et al., 1986; Korn et al., 1987) and suggests a functional connection between signaling pathways involving tyrosine kinases and ras proteins. Moreover, the recent discovery that the product of the neurofibromatosis type 1 gene (NFI) exhibits ~21” GAP activity (Martin et al., 1990; Xu et al., 1990; Ballester et al., 1990) is likely to help define the signaling defect underlying this pathology. Proteins that stimulate the GTPase activity of three other low molecular mass GTPases have also been reported.

Protein

GAPS specific for the rho and rab3A gene products were identified in spleen (Garret et al., 1989) and brain (Burstein et al., 1991). Two cytosolic GAPS specific for p21raPap’have been identified in bovine brain (Kikuchi et al., 1989), and a membrane-associated raplGAP has been purified to near homogeneity from HL60 cells (Polakis et al., 1991). A presumed function of these raplGAPs is the downregulation of ~21”’ activity. Although the function of P21 raP’ itself is unclear, some studies suggest that it may act to antagonize the action of ~21”“: a cDNA encoding p21raP1 reverts transformation when expressed in Kirsten ras-transformed NIH 3T3 cells (Kitayama et al., 1989). p21faP1has also been reported to inhibit ras-induced germinal vesicle breakdown when microinjected into oocytes (Campa et al., 1991). By analogy with other GTP-binding proteins, p21raP’ probably requires bound GTP for functional activity. Point mutations analogous to those that either increase or decrease the transforming activity of ~21”” result in rap1 proteins with the corresponding alterations in transformation suppressor activity (Kitayama et al., 1990). Although the mechanism of suppression is not understood, it is known that p21raP1 binds to rasGAP in vitro in a GTPdependent manner, without affecting p21”P’ GTPase activity (Frech et al., 1990; Hata et al., 1990). Considering that rasGAP or GAP-related proteins may be downstream effecters for ~21”” (McCormick, 1989; Hall, 1990b), it is possible that binding of p21 rap7to rasGAP may account for its ability to interfere with ras effector functions (Frech et al., 1990). In addition to antagonizing the effects of ras, ~21”~’ has been implicated in the CAMP-mediated inhibition of platelet metabolism (Kawata et al., 1989; Lapetina et al., 1989) and appears to be identical to thrombolamban (Fischer and White, 1987), a major substrate for protein kinase A in these cells. Phosphorylation of p21rEp1 in response to hormones that elevate intracellular CAMP correlates with translocation of p21 rap’ from a membrane to a cytosolic fraction (Kawata et al., 1989; Nagataet al., 1989; Lapetina et al., 1989). On the other hand, agents known to activate platelets promote translocation of p21raP’ from a cytosolic to a cytoskeletal fraction (Fischer et al., 1990). p21”P’ was also identified as a GTP-binding protein tightly associated with cytochrome b purified from human neutrophils (Quinn et al., 1989), suggesting its involvement in the inflammatory response of these cells. To better understand the cellular activites of p21”P’, we sought to isolate and characterize a key regulatory protein that specifically stimulates its GTP hydrolytic activity. In this article we report the molecular cloning and expression of a cDNA encoding a GAP specific for ~21’~“‘. Results Cloning of raplGAP cDNA raplGAP was purified from bovine brain membranes

(see

Cell 1034

A GGCCGCGGGCACCAGAGTGCCGAGCCCAGGACGCCCCCGGCCCAGGCCCTTGGGGTGGAC~GTCCTTCACTTCTCGCCGGAGTGTGTGGAGGAGCGATGGGCAG~CCAGCACTTCCCT CAGGCACTAGACCTGTCACGAGTGAACTTAGTTCCCTCCTATACTCCTTCACTCTACCCTAAGAACACAGAT MIEKMQGSRMDEQ

-82 39 13

CGCTGCTCCTTCCCGCCGCCCCTCAAAACAGAGGAGGACTACATTCCATACCCGAGCGTGCACGAGGTCTTGGG~GAG~GGACCCTTCCCCCTCATCCTGCTGCCCCAGTTTGGGGGC RC S F P P P L K T E ED Y I P Y P S V H E V L G RE G P F P L I L L P 0 F G G

159 53 219 93

TTTCTCGGCAAGGAGCATTTCAATTACTACTACTCACTGGACACTGCCCTCGGCCACCTTGTCTTCTCACTC~GTACGATGTCATCGG~ACC~GAGCACCTGCGGCTGCTGCTCAGGACC FLGKEHFNYYSLDTALGHLVFSLKYDVIGDQEHLRLLLRT

399 133

AAGTGCCGGACATACCATGATGTCATCCCCATCTCCTGCCTCACCGAGTTCCCTAATGTTGTCCAGATGGCARAGTTGGTTGGTGTGTG~GACGTC~TGTGGATCGGTTCTATCCTGTGCTC KCRTYHDVIPISCLTEFPNVVQMAKLVCEDVNVDRFYPVL

519 173

TACCCCAAGGCTTCCCGGCTCATCGTCACCTTTGACGAGCATGTCATCAGC~~~CTTC~GTTTGGCGTCATTTATCAG~GCTT~~GACCTCCGAGG~G~CTCTTCAGCACC YPKASRLIVTFDEHVISNNFKFGVIYaKLGQTSEEELFST

639 213

AATGAGGAAAGTCCCGCTTTCGTGGAGTTCCTTGAATTTCTTGGCCAGAAGGTCAAACTGCAGGACTGCAGGACTTT~GGGGTTCCGAGGA~CCTGGACGTGACCCACGGGCAGACGGGGACCG~ 759 NEESPAF~EFLEFLGQKVKLQDFKGFRGGLDVTHGQTGTE 253 TCTGTGTACTGCRACTTCCGCAACAAGGAGATCATGTTTCACGTGTCCAC~GCTGCCATACACGG~GGGGACGCCCAGCAGTTGCAGCGG~GCGGCACATCGG~CGACATCGTG S 'I Y C N F RN KE I M F H V S T KL EY T E G DAQ Q LQ R K RH I G N D I"

879 293 939 333

GTCTCTGTCACTGCAAGAGATGATGTGCCCTTCTTTGGACCCCCCCTCCCGGACCCCGCTGTGTTCAGG~GGGGCCTGAGTTC~~~TTTTTGCTGAC~GCTGATC~TGCTG~ VSVTARDDVP FFGPPLPDPAVFRKGPEFQEFLLTKLINAE

1119 373

TATGCCTGCTACAAGGCAGAGAAGTTTTGCC~CTGGAGGAGCGGACGCGGGCCGCCCTCCTGGAGACGCTCTATGA~~CTA~CATCCACAGCCA~rCCATGATGGGCTTGGGCGGC YACYKAEKFAKLEERTRAALLETLYEELHIHSQSMMGLGG

1239 413 1359 453 1479 493

GGCTCCCGCCGCAGCAGCGCCATTGGCATCGAG~CATACAGGAGGTGCAGGAG~GAGGGAGAGCCCTCCGGCTGGTCAG~GACCCCAGACAGC~GCACGTCTCACAGGAGCCC~G GSRRSSAIGIENIQEVQEKRESPPAGQKTPDSGHVSQEPK

1599 533

TCGGAGAACTCATCCACTCAGAGCTCCCCAGAGATGCCCACGACC~G~CAGAGCGGAGACCGCAGCGCAGAGAGCAGAGGCGCTC~GGACTTCTCCCGCTCCTCGTCCAGTGCCAGC SE@jSSTQSSPEMPT TKNRAETAAQRAEALKDFSRSSSSAS

1719 513

AGCTTCGCCAGCGTGGTGGAGGAGACGGAGGGTGTGGACGGAGAGGACACAGGCCTGGAGAGCGTGTCATCCTCAGG~CACCCCAC~GC~GACTCCTTCATCTATAGCACGTGGCTG SFASVVEETEGVDGEDTGLESVSSSGTPHKRDSFIYSTWL

1839 613

GAGGACAGTGTCAGCACCACTAGTGGGGGCAGCTCCCCAGGCCCCTCTCGATCACCCCACCCAGACGCCGGC~GTTGGGGGACCCTGCGTGTCCCGAGATC~GATCCAGCTGG~GCA EDSVS? TSGGSSPGPSRSPHPDAGKLGDPACPEIKIQLEA

1959 653 2079 663

GACCTCTGCCCTGAAGACCACACCAGCCCGTGGGCT~CCCCTGCCTCCCCACCCTCCCCATGGCCCACCCATCTGGGCTGTCTCTGCAGGGCAGAGCCGTCCAGACCTGGGATCAGGG 2199 AAGCTGCTGGCATCGTCCCCACCCCCAGCCTGGGGGTCTGCTGGGGCAGGGATTGCTCAGTGG~GCAGGACTGGGGGTCTGGCTTGCCCCCTCCCTGGGCCTCCATCACCCCTGAGC 2319 ATCCCTCTGGACTCAGAGGGAACAAGGTGGGAGAGAGAGTTTGAGACAGCTCCGTGTGGAGAGCTTAGCCCCTGGAGGCAGCAC~GGAGGATGTGATATGTGGGGGAGTGAGCACTGGG 2439 TTGGGAGCCGGGTCCTGGTTTCCAATTTGGGTTCTGCTGTGTGACTCTGGGCAAGTCACTCTCCCTCTCTGGGCATGTCTGCTAC~TGGAC~GATTATTTCAGAGGTCACTG~GAC 2559 TGTGATTACATGCACCTGCCTTAGAAGGTAGGTAGGATTTTCTTCCCAGGGACCTCCTATCACCCTACCCTGCTTCTTGAGGTCCCTGGAGCCCCAGGTGGGCTGAGGGGCAGGGAGCCGGCTG 2673 TGCCCAGTATGCCTCCTGGACCCTCCAGTTCTGCCACAGGTCTGCCGATGCCCTGTCCACTGCCTACACATGACAGAC~GT~CCCCCTCATGGGGGATGGGGACCTACCTGGCTCCTC 2799 AGCCAGCACCCAGCTTAACCCCTGCCATCCCATGCTG~CCCTCCAGGCC~GAGTCTCAGCTGGCCGAGAGTCCAGGCCTTGCCTCCCCGACCGCCATGGAGGGGGCAGCCCGGCACAG 2919 CTGCTGGGAGCCCTTGTGTGTCTGGTCACACTTTTTAGGCGTCACGCC~GGCCAGCCTCCTGGCCCC~TACCCATTTTGG~GCCCCTGTGGCCGTGTGGATGTCGGT~CAGTTGT 3039 ATYTAAATTCTATTTATCGCTATTGT3097

B SLLIPGKSASR..... FG.RRGSAIGIGTVEE I I /I/I I II II I I I I I SLIVPGKSPTRKKSGPFGSRRSSAIGIENIQE Figure

I.

Nucleotide

and Deduced

Amino

Acid Sequence

I of rap1 GAP cDNAs

(A) The HuBlO-A nucleotide sequence is presented with the predicted amino acid sequence (single letter code), beginning at the first initiation codon. Peptides generated from CNSr digestion of raplGAP purified from bovine brain are underlined. Putative N-linked glycosylation sites are circled. The arrowhead indicates the 5’ and 3’ ends of HP3-12 cDNA. The solid diamond indicates the location of the following additional 78 base sequence contained in the HP3-12 cDNA: TTGCTTATTCCTGGGAAAAGTGCGAGTAGATTCGGACGCCGGG GCAGTGCCATAGCATAGGAACCGTGGAAG AGTCA. (6) The amino acid sequence predicted from the additional 78 nucleotide bases in the HP3-12 cDNA shown in comparison with the amino acid sequence immediately following it and contained in the HuBlO-A amino acid sequence.

the Experimental Procedures); a prominent 88 kd polypeptide that comigrated with the GAP activity was detected on the final step of column chromatography. Partial amino acid sequence was obtained from two CNBr fragments, and synthetic oligonucleotides based on the most probable sequences encoding these peptides were used to screen human brain and placental hgtll cDNA libraries.

One cDNA clone, designated HuBlO-A, encoded a predieted polypeptide of 883 amino acids with a calculated molecular mass of 73.4 kd and contained sequences corresponding to both CNBr peptide fragments (Figure 1A). The 3’ sequence terminated with a polyadenylation tract, and the 5’ sequence contained a translation initiation site, complying with Kozak’s criteria (Kozak, 1984). The amino

Cloning

of raplGAP

1035

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2. Expression

49.5-

of rapGAP

cDNAs

in Insect Sf9 Ceils

(A) Chromatography of recombinant raplGAP. A lysate prepared from Sf9 cells overexpressing the HP3-12 cDNA in the pAcRG4 expression vector was chromatographed on S-Sepharose. raplGAP activity is expressed as percent GTP hydrolyzed. (6) SDS-PAGE of S-Sepharose chromatography fractions. One microliter of lysate (L) or flow through (FT) or 3 nl of each fraction was applied to the gel. Protein was stained using Coomassie blue. Values at left are molecular weights (x lo-‘) of standard proteins (S). (C) Western blot. Sf9 cells expressing the HuBlO-A cDNA in the pAcRG9 expression vector were lysed, and membrane and cytosolic fractions were prepared in equal volumes. raplGAP immunoblotting was with anti-GQT. Twenty microliters of membrane suspension (M), 1 nl of cytosol (C), or 200 ng of partially purifed bovine brain raplGAP (BovBr) was applied to the gel.

acid sequence did not exhibit significant homology to any known sequences contained in the NBRF data base. In particular, no similarity with rasGAP, NFl, or IRA 1 was detected. Asecond cDNA, designated HP3-12, was identified from human placenta; this partial cDNA contains sequence identical to HuBlO-A but also encodes a duplicated amino acid sequence consisting of 26 residues in the carboxy-terminal region of the protein (Figure 1 B). This duplicated sequence cannot be found in other proteins in the current data bases. Three putative N-linked glycosylation sites were identified in the deduced raplGAP sequence, but there is no apparent signal sequence expected for translocation of the nascent polypeptide into the endoplasmic reticulum. A hydrophobicity analysis did not reveal any regions particularly rich in hydrophobic residues (data not shown), suggesting that raplGAP, which was purified from cell membranes, may be anchored to cell membranes through association with other cellular components rather than thrqugh transmembrane sequences. Expression of raplGAP cDNA raplGAP cDNAs were expressed using the AcNPV-Sf9 baculovirus expression system. raplGAP activity was ap-

proximately lO,OOO-fold higher in lysates from cells infected with the HP3-12 or HuBlO-A recombinant virus, compared with lysates from Sf9 cells infected with virus containing deleted polyhedrin promoter only. Lysates from Sf9 cells expressing the HP3-12 cDNA contained a protein with an apparent molecular mass of 75-85 kd that accounted for approximately 50% of the total lysate protein (Figure 28, lane L). This diffuse staining pattern appears to be composed of several bands that were partially resolved and comigrated with the GAP activity when chromatographed on an S-Sepharose column (Figure 2A). The high yield of rap1 GAP protein from the infected Sf9 cell permitted the purification to near homogeneity of approximately 7 mg of raplGAP from 100 ml of Sf9 cell suspension. A synthetic peptide corresponding to an internal amino acid sequence of the predicted raplGAP polypeptide was used for the generation and affinity purification of rabbit antibodies specific for raplGAP. This antibody reacted strongly on Western blots, with an 85-95 kd protein present in lysates from Sf9 cells overexpressing the H&IO-A cDNA (Figure 2C). These same immunoreactive bands were also detected in the membrane fraction from the recombinant Sf9 cell, although this fraction contained only ~5% of the total recovered immunoreactivity. The antibody also recognized the raplGAP partially purified from bovine brain membranes (Figure 2C). Biochemical Properties of raplGAP There are two closely related rap1 proteins, p21faprA and P21 r*@, that are 95% identical (Pizon et al., 1988a). The raplGAP characterized in this study stimulated both of these proteins with approximately equal sensitivity. Numerous other GTPases share sequence similarities with raplA and rap1 B gene products (reviewed in Sanders, 1990). The closest known mammalian relative is rap2 (Pizon et al., 1988b), which encodes a protein 63% identical to p21raP7,whereas the N-ras and rabl and 3 gene products are approximately 53% and 34% identical to p21rapap’,respectively (Touchot et al., 1987). To demonstrate the specificity of the recombinant rap1 GAP, we tested its ability to stimulate the GTPase activities of these proteins. We used a concentration of raplGAP IO-fold higher than that required for complete hydrolysis of GTP bound to p21r8p’. No stimulation of the ras or rab proteins was observed (Figure 3A). p21’@ was weakly responsive to raplGAP, but the degree of stimulation indicated that it was at least lOO-fold less sensitive than p21 @‘l. We also compared the specific activities of rap1 GAP and rasGAP toward their respective GTP-binding protein substrates. Both GAPS exhibited specific activities of approximately 50,000 pmollminlmg. The binding affinity of p21raP’ for rap1 GAP was also measured. A dissociation constant was estimated by determining the concentration of p21 raP1required to inhibit raplGAP activity toward a trace quantity of [r-3ZP]GTP-labeled p21mP’. When liganded to the nonhydrolyzable GTP analog, GppNHp, p21’@ produced half-maximal inhibition at a concentration of 30 to 35 uM (Figure 3B). However, in contrast to the lack of binding reported for p21reS-GDP to rasGAP (Vogel et al., 1988), p21rBP1-GDP effectively inhibited raplGAP at concentrations similar to those required

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3. Specificity

and Binding

of Purified

Recombinant

raplGAP

(A) Specificity. Each of the indicated proteins (2 pmol) was prebound to [T-“P]GTP and incubated with 20 ng raplGAP for 5 min at 25%. (6) Competition binding. The indicated concentrations of p21”p’ prebound to GDP or GppNHp were included in the raplGAP assay. “Control” indicates the addition of p21 ‘W buffer-equivalent containing the appropriate concentration of GppNHp. The result presented is typical of three separate experiments.

for the GppNHp-bound p2irap7 (Figure 3B). p21raP1has also been reported to bind to rasGAP with high affinity (Frech et al., 1990). We therefore tested the reciprocal situation but detected no significant association of ~21”” with raplGAP (data not shown). The expression of the cDNAs encoding rap1 GAP consistently resulted in the production of a set of four or five protein bands associated with the GAP activity. This was observed with the Sf9 cell baculovirus system (Figure 28) as well as with transient expression in human 293 cells (data not shown). To determine whether the multiple forms resulted from internal modification or degradation of the protein, Sf9 cells expressing rap1 GAP were pulse-chase labeled with [35S]methionine, and the fate of the translated rap1 GAP was monitored by SDS-polyacrylamide gel electrophoresis. raplGAP underwent incremental increases in apparent molecular mass following its synthesis in the Sf9cell (Figure4A). Because some posttranslational modifications proceed through a preprocessing step, an experiment was carried out to determine whether amino- or carboxy-terminal residues were removed during rap1 GAP

A

B=I0 c

0

30'

60'

120'

360'

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24hr

3

processing. cDNAs encoding raplGAP proteins with an additional 9 amino acid polypeptide epitope at either the amino- or carboxy-terminal end were expressed in Sf9 cells. Antibodies specifically reactive to this epitope recognized all of the bands associated with the expressed raplGAP chimeric proteins (Figure 48). These experiments demonstrate that the multiple forms of the expressed raplGAP are due to a posttranslational modification of the intact polypeptide chain. The nature or site of this modification is unclear. Exhaustive treatment of the protein with acid and alkaline phosphatases had no effect on the appearance of the multiple bands (data not shown). Treatment with N-Glycanase, under conditions that produced a shift in the molecular mass of the glycosylated c-erbB2 protein, had no effect on raplGAP (data not shown). In addition, recombinant Sf9 cells expressing either raplGAP or the c-erbB2 gene product were incubated with tunicamycin, an inhibitor of protein glycosylation. This treatment resulted in a c-erbB2 protein with increased mobility on SDS-polyacrylamide gels, but again there was no affect on rap1 GAP (data not shown).

g2, 29;;

Figure 4. Posttranslational rap1 GAP

Modification

of

(A) Pulse-chase. The HuBIO-A cDNA was axpressed in Sf9 cells pulsed with [%]methionine and then chased with excess unlabeled methionine at the indicated times. “c” indicates uninfected Sf9 cells. Values at left are molecular weights (x 10e3). (B) Immunoprecipitation. HP3-12 cDNA was modified to express raplGAP fusion proteins containing the 9-mer “Glu-Glu” epitope at the amino (N-Glu) or carboxyl (C-Glu) terminus. The expressed proteins were immunoprecipitated with anti-“Glu-Glu.” rGAP is 1 ug of purified recombinant HP3-12 raplGAP. Protein was stained using Coomassie blue.

Cloning 1037

of raplGAP

ADULT

Figure

5. lmmunoblot

Analysis

of Tissue

Fractions

FETAL

and Cell Lines

All blots were developed using rabbit antisera raised against purified recombinant HP3-12 raplGAP. Except where indicated, was applied to each lane. (A) Cell lysates. Cell lines are described in the Experimental Procedures. “d-HLGO” indicates differentiated HL60. “HuBlO-A” from Sf9 cells expressing this raplGAP cDNA. “bov br” is 100 ng of total protein of rap1 GAP partially purified from bovine brain is 10 ng of purified HP-3-12 raplGAP. (B) Membrane fractions from adult rat tissues. (C) Cytosolic extracts from human adult and fetal tissues. At left are molecular weights (x IO-$ and arrow at right indicates

Based on these results, it is unlikely that N-linked glycosylation is responsible for the multiple forms of raplGAP. Tissue Distribution of raplGAP To examine the tissue distribution of raplGAP protein, a rabbit polyclonal antibody was raised against recombinant raplGAP purified from Sf9 cells overexpressing the HP312 cDNA. Western blot analysis of crude membrane fractions prepared from several different adult rat tissues indicated that only brain membranes contained substantial levels of an 85-95 kd immunoreactive polypeptide (Figure 5B). This immunoreactive protein was also present at relatively high levels in lysates prepared from the Wilms’ kidney tumor cell line SK-NEP-1 but was much less abundant or absent in lysates from a second Wilms’ tumor cell line, G-401, and the 293 kidney cell line (Figure 5A). NIH 3T3 cells, either untransformed or transformed by v-sfc or oncogenic H-ras, did not contain detectable levels of immunoreactivity (Figure 5A). The HL60 promyelocytic leukemia cell contained significant amounts of the 85-95 kd polypeptide. However, this reactivity was dramatically reduced when the cells were induced to differentiate into granulocyte-like cells (Figure 5A). The selective presence of raplGAP in SK-NEP-1 and undifferentiated HL60 cells suggested that it may be involved in the programming of cell growth and division. We therefore carried out comparative Western blotting analysis of human adult and fetal tissue extracts. Fetal liver, lung, and brain all expressed much higher levels of raplGAP than the corresponding adult tissues (Figure 5C). We also estimated the specific activity of raplGAP in many of the samples analyzed by Western blotting. Surprisingly, many of the sources examined contained high levels of raplGAP activity, even though the 85-95 kd immunoreactive band was not detected (compare Figures 5 and 6). For example, spleen membranes exhibited the

50 ug of total protein is 50 ng of cell lysate membranes. HP3-12

position

of rap1 GAP.

highest level of rap1 GAP activity, yet no immunoreactivity was detected. Most striking was the relative increase in raplGAP activity following differentiation of the HL60 cells, even though the 85-95 kd immunoreactive protein decreased. This suggests that an immunologically distinct raplGAP is present in many of the sources tested here. Northern blotting analysis resulted in the identification of a single 3.3 kb transcript in mRNA derived from several immortalized cell lines (Figure 7A). The highest levels of expression were detected in cell lines derived from human malignant melanoma, SK-MEL-3, and the Wilms’ kidney tumor, SK-NEP-1. In agreement with the Western blotting results, the 293 and G401 kidney cell lines contained relatively low or undetectable levels of rap1 GAP mRNA. Detectable levels of the 3.3 kb mRNA were also found in cell lines derived from breast (MCF-7) adrenal (Genoa), and

rFigure

ceil Ipates

6. Distribution

1

of RaplGAP

L

t

membranes

JLCI~GAPI

Activity

Six micrograms of total protein from the indicated cell lysates or rat membrane fractions was assayed for raplGAP activity. “I”, “3”, and “6” refer to ng of purified HP3-12 raplGAP.

Cell 1038

Figure

7. Northern

Blot Analysis

(A) Cellknes. Poly(A)t mRNA (4 kg) from each of the indicated sources was applied to the gel. (B) Comparison of raplGAP mRNA and cDNA. HuBlO-A cDNA was excised from the lambda ZAP vector using EcoRI, denatured with NaOHl EDTA, and coelectrophoresed with mRNA from SK-NEP-1 ceils. Marker positions are indicated by values ‘(kb) at right.

ovarian (Ovcar-4) carcinomas. On longer exposures, lower levels of mRNA could be seen in several other tumor cell lines as well. The denatured HuBlO-A cDNA was also applied to the gel and found to comigrate exactly with the 3.3 kb mRNA from the SK-NEP-1 cell line (Figure 78). This is consistent with the comigration of the raplGAP protein from SK-NEP-1 cells with the product of the HuBlO-A cONA (Figure 5A) and demonstrates that the HuBlO-A cDNA represents a complete complement of the rap1 GAP mRNA. Due to an alternative mRNA splicing event, the HP3-12 cDNA encodes a rap1 GAP containing a duplicated amino acid sequence, of which only one copy is found in the product of the HuBlO-A cDNA (Figure 1). We examined the relative prevalence of the two mRNAs in several tissues and cell lines. Polymerase chain reaction (PCR) products were generated from mRNA using specific primers bordering the region of the duplicated sequence. The mRNA from all of the cell lines described in Figure 7A generated only a single PCR product of the size expected from the HuBlO-A cDNA (data not shown). Several other cell lines also contained only the HuBl O-A sequence; however, a second PCR fragment, of the size expected from HP3-12 cDNA, was generated from mRNA extracted from adult brain (Figure 8, left panel). Although much fainter, a band of this size was also generated from placenta and fetal brain mRNA. Additional larger products were also derived from adult brain. To verify that the larger PCR products generated from adult brain actually contained the additional 78 base pairs unique to HP3-12, we blotted the

fragments and probed them with an oligonucleotide containing sequence specific to this region. As expected, the fragment generated from HP3-12 was positive and that from HuBlO-A negative (Figure 8, right panel). The fragments derived from adult brain, one equivalent in size to that from HP3-12 and a second one larger than this, were also strongly positive (Figure 88, right panel). Products from placenta, fetal kidney, and fetal brain also reacted but were present at much lower levels. These results show that the mRNA splicing variant represented by the HP3-12 cDNA is expressed in only a subset of tissues and that its expression in brain may be activated at a given stage of development. Discussion In this study we have determined the primary structure of a GTPase activating protein specific for p21”P’. To our surprise it shows no significant homology to the rasGAPs, even though p21”“and p21 rap7are 53% identical (Pizon et al., 1988a). A comparison of the amino acid sequences of rasGAP, the NFl gene product, and the yeast rasGAPs, IRA1 and 2, suggests that certain conserved amino acids are essential for the GTPase activating function of these proteins (Ballester et al., 1990). None of these conserved regions could be identified in rap1 GAP. rap1 GAP and rasGAP differ in two other important aspects: First, they appear to utilize different mechanisms to bring about GTP hydrolysis. For p21ras, glutamineis essential for GAPmediated GTP hydrolysis. p21ranap’contains threonine at

Figure 8. Discrimination 10-A mRNAs by PCR

of HP3-12

and HuB-

HP3-12 or HuBlO-A cDNA or 50 ng each of poly(A)+ mRNA from each of the indicated sources was used as a template for the PCR reactions, (-) indicates no mRNA. The left panel shows agarose gel of PCR products. The right panel shows Southern blot of PCR fragments using a probe specific to sequence in the HP3-12 cDNA.

Cloning 1039

of raplGAP

this position; replacement of threonine by glutamine does not affect raplGAP-mediated GTP hydrolysis (Haubruck et al., 1991), indicating that this residue is not critical for this reaction. Second, binding of ~21”” and p21raP’ to rasGAP is GTP dependent. Binding of p21raP7 to its GAP, on the other hand, occurs with both GTP- and GDP-bound forms. The ability of the raplGAP to associate with the cell membrane may relate to a duplicated 26 amino acid sequence found in the carboxy-terminal region of the HP3-12 rap1 GAP. This sequence is represented only once in the HuBlO-A raplGAP. The two recombinant proteins exhibit similar specific activities, suggesting that this carboxyterminal portion of the protein may be important for functions other than raplGAP activity. We found that adult brain was the only tissue source that contained significant levels of immunoreactive rap1 GAP in the membrane fraction. This tissue also contained the most abundant levels of the mRNA encoding a raplGAP with the duplicated amino acid sequence. Thus, it is possible that the duplicated region may, in some way, provide additional avidity required for interaction at the membrane. The rap1 GAP cloned in this study is not expressed ubiquitously. Western and Northern blotting analysis indicated that the most abundant sources were fetal tissues and undifferentiated cells. This distribution suggests that the raplGAP may function in the propagation of growthpromoting signals. Following differentiation of the HL60 cell, the raplGAP immunoreactivity was reduced to an almost undetectable level. This modulation may be part of the developmental program that is engaged during the maturation of progenitor cell types. However, the functional outcome of losing this rap1 GAP during maturation of the cell cannot simply be due to a loss of overall raplGAP activity. In fact, the differentiated HL60 cell exhibited higher levels of activity than the undifferentiated cell, suggesting the presence of an immunologically distinct raplGAP. This is not surprising, as our laboratory and others have previously noted the presence of chromatographitally distinct raplGAP species from HL60 cells (Polakis et al., 1991) and bovine brain (Kikuchi et al., 1969). The specific loss of the 85-95 kd raplGAP protein following differentiation of the HL60cell, without aconcurrent loss of total rap1 GAP activity, suggests that specialized functions may be maintained by the different raplGAPs. These unique functions might include the capacity to localize to different subcellular compartments or to associate with different proteins. It is also conceivable that distinct raplGAPS possess unique catalytic or regulatory properties that are activated through their binding to p21”P’. The unique ability of p21 raP’ to associate with both rasGAP and raplGAP suggests that its functional output in vivo may be dependent upon its interaction with these two proteins (Figure 9). On binding to rasGAP, p21”P’ may antagonize the interaction between ~21”” and rasGAP, as has, been demonstrated in vitro (Frech et al., 1990; Hata et al., 1990). On the other hand, binding to rap1 GAP results in the conversion of p21raP’ from the GTP- to the GDP-bound state. Because the binding of p21raP7 to rasGAP is GTP dependent (Frech et al., 1990) the ability of p21raP’ to an-

(nonDroductivel

GAP

p21 rap1 -GDP

qi21rapliTP

~pPlrapl.GTP pi

p2lraSeGTP

(pkductive)

GRCJWTH Figure 9. Schematic Representation of the Proposed between p21@, p21ras, and Their GAPS

Interactions

tagonize rasGAP would be regulated by its interaction with its own GAP. This interplay between p21rap1 and the two GAPS may serve to buffer the effective growth-promoting activities of ras. In this scenario, binding of p21ras to rasGAP, or alternatively, to adistinct effectortarget, isconsidered to result in a positive growth signal (Hall, 1990b; McCormick, 1989). This signal would be disrupted by the binding of p21@-GTP to the same downstream effector. Under conditions of growth, raplGAP would be expected to maintain p21raP’ in the GDP-bound state, where it would not function effectively as an antagonist, thus allowing ~21” free access to its downstream target. Alternatively, ~21’~“’ may not directly compete with the target for ~21’~~ but rather signal antagonistically through a separate pathway that nevertheless requires the GTP-bound form of ~21”“‘. Either mechanism is consistent with the observation that rap1 suppresses the ras-transformed phenotype (Kitayama et al., 1989). Accordingly, the down-regulation of P21’@ by raplGAP would be expected to contribute to cell growth. This is in agreement with our finding that raplGAP is relatively more abundant in dividing undifferentiated cells. The vast array of ‘recently discovered ras-related GTPbinding proteinssuggeststhat these molecules participate in numerous and diverse signaling systems in the cell. Exocytosis (Fischer von Mollard et al., 1991), endocytosis (Chavrier et al., 1990), axonal transport (Bielinski et al., 1989),cytoskeletalassembly(Chardinet al., 1989),vesicle trafficking (Balch, 1990) and cell growth (Korn et al., 1987; Mulcahy et al., 1985) all appear to be dependent upon these low molecular mass GTP-binding proteins. It is not yet clear whether each of these GTP-binding proteins is regulated by its own GTPase activating protein; however, several new GAPS have recently appeared on the horizon. Based on the lack of structural similarity between rasGAP and rap1 GAP, we may now expect these new GAPS to be entirely different proteins. The anticipated cloning of GAPS

Cell 1040

specific for ral, rho, rab3A, yptl , and other ras-related proteins may ultimately allow for their categorization and lead to a more integrated model of their functions in cell signaling. Experimental

Procedures

Purification of raplGAP For the purification of raplGAP from bovine brain, crude membranes were prepared essentially as described previously (Waldo et al., 1987). Detergent extraction and column chromatographies were carried out as described previously for the purification from HL60 cell membranes (Polakis et al., 1991), except that Sephacryl S-300 chromatography was substituted for the hydroxyapatite step, and a final chromatography on a cation exchange SPd-PW HPLC column was added. Digestion of purified protein with CNBr and isolation of peptide fragments for amino acid sequencing were carried out as described previously (Polakis et al., 1969). For the purification of recombinant raplGAP, Sf9 cells overexpressing the HP3-12 cDNA (see below) were lysed by freeze-thawing in 20 mM Tris (pH 6.0), 1 mM dithiothreitol (DTT), 1mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, leupeptin, and pepstatin at 1 pglml. Following centrifugation at 100,000 x g for 1 hr, the supernatant was adjusted to pH 6.5 and chromatographed on S-Sepharose essentially as described above for bovine brain extracts, except that the detergent was omitted. Isolation of cDNA Clones Amino acid sequences of two peptides were obtained from CNBr digestion of bovine brain raplGAP. 7Ka: lASNFL(P)AYlVVQAENPGTEPP(A)YK and 2.5K: F(G)VSTKLPFT. Subsequent digestions yielded another peptide sequence similar to 7Ka, 7Kb: IASNFLSAYVVVQAEGGGPDG(X)LYKV. Based on the amino acid sequence of a portion of the 7Ka peptide (AYIVVQAENPGTEPP) and codon usage statistics (Lathe, 1965), aconsensusoligonucleotidewith thesequence5’-GGGGGCTCTGTGCCAGGGTTCTCAGCCTGCACCACAATGTAGG C-3’ (BR67) was synthesized. This oligonucleotide was radioactively labeled with T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and [Y-~~P]ATP (Amersham Corp., Arlington Heights, IL) and initially used to screen a human placental cDNA library (2.5 x 1O5 clones) in hgtll (Clonetech, Palo Alto, CA). Duplicate nitrocellulose filters of recombinant plaques were hybridized as follows: first lift under high stringencyconditions(16 hr at 42°C with 50% formamide in hybridization buffer [5x SSC, 5 x Denhardt’s solution, 50 pg/ml salmon sperm DNA, 50 mM sodium phosphate, 0.1% SDS]) and second lift under low stringency conditions (16 hr at 42OC with 30% formamide in hybridization buffer). The filters were washed with 2 x SSC, 0.1% SDS and 0.2 x SSC, 0.1% SDS at 65OC. One hundred and six positive clones were identified, of which 36 were pursued for further characterization. Positive recombinant bacteriophages were purified, cDNA inserts were digested with EcoRl and subcloned into pGEM (Promega, Madison, WI), and nucleotide sequence was determined by primed DNA synthesis in the presence of dideoxynucleotide triphosphates (Sanger et al., 1977; Messing et al., 1981). The sequence obtained from one of these clones, HP3-12, was used to generate a perfect matched oligonucleotide, with the sequence 5’-ACCAAGGTGAAGCTCGAGTGCAACC-3’(BR115) or a radioactively labeled 233 bp or 278 bp product generated by the PCR. These probes were used to rescreen the Clonetech placental library (1 x IO8 clones) or a human fetal brain library (1 x IO6 clones) in lambda ZAP (Stratagene, La Jolla, CA). Duplicate filters were hybridized and washed as described above. The radioactively labeled PCR product was generated as follows: l-10 ng HP3-12 cDNA EcoRl fragment was used as template, along with 2 pM each dATP and dTTP, 1 PM each dCTP and dGTP, 1 FM each (100 PCi) [@P]dCTP and [a-3ZP]dGTP, 10 pmol each of upstream (BR119 5”AAGCACTTTCTCGGCAAGGA-3’ or BRI 15) and downstream primer (BR120 5”GAACCGATCCACATTGACGT-31, and 1 U Taq poly merase. The reaction cycle was 95“C for 30 s, 63°C for 30 s, 72OC for 30 s; 20-25 cycles. The resulting product was purified through a spin column (Bio-Rad, Richmond, CA). Five positive clones were obtained from screening of the Stratagene library, of which four were identical. The lambda ZAP plasmid containing the cDNA was liberated as described by Stratagene. Sequencing of one of these clones, HuBlO-A, yielded 5’ and 3’ sequences that extended beyond those of HP3-12.

Expression of raplGAP cDNAs in Insect Sf9 Cells The rapGAP cDNAs were recombined into the Autographa californica baculovirus, using transfer vectors constructed by insertion of the rapGAP-encoding restriction fragments into pAcCl3, a transfer vector described previously (Munemitsu et al., 1990). The transfer vector pAcRG4 was constructed by insertion of the EcoRl fragment from HP3-12 into the Kpnl-BamHI site of pAcC13, via the Kpn-EcoRI 5’ adaptor containing a nucleotide sequence encoding a consensusinitiating methionineand the”Glu-Glu”antibodyepitope(Grussenmyer et al., 1985). The transfer vector pAcRG9 was constructed by insertion of the EcoRl fragment from HuBlOA into the EcoRl site of pAcC13. To generate recombinant virus, 2 Kg of the appropriate pAc transfer vector was cotransfected with 1 Kg of wild-type viral DNA into Sf9 cells as previously described (Summers and Smith, 1987). Recombinant virus was isolated by plaque purification (Smith et al., 1983). To produce recombinant raplGAP, 1 x IO6 to 1.5 x 10’ cells per ml were infected with 5-10 PFU of recombinant virus per cell. Suspension cultures were grown in stirred flasks containing protein-free medium (Miaorella et al., 1988) and were harvested 48 hr post infection. For pulse-chase experiments, seven 60 mm dishes of confluent Sf9 cells were infected with either the raplGAP recombinant baculovirus vector AcRG9 or with control vector containing a deleted polyhedrin promoter region. At 18 hr post infection, cells were starved for 30 min at 25OC in Grace’s methionine-free medium including 10% dialyzed fetal calf serum and then labeled with 250 VCi [35S]methionine for 20 min at 25°C. The cells were rinsed twice and either harvested immediately or incubated with growth medium for the indicated times and harvested thereafter. raplGAP Assays For the detection of raplGAP activity, we used the nitrocellulose filter binding format described previously (Polakis et al., 1991), except in thecompetition binding and specificityexperiments(Figure3), in which the phosphate release assay (Halenbeck et al., 1990) was used. GAP activity was expressed as the percent GTP bound to p21ra@ that was hydrolyzed relative to buffer control.

Antibodies Immunization procedures for the production of rabbit antibodies were carried out by Berkeley Antibody Co., (Richmond, CA). The rabbit anti-GQT serum was raised against the synthetic peptide corresponding to the sequence GQTSEEELFSTNEES (residues 203-217) contained in the predicted primary structure of raplGAP. The antibodies were affinity purified against the immobilized peptide using CNBractivated Sepharose (Pharmacia, Inc.) according to the manufacturer’s instructions. Antiserum raised against the purified recombinant HP312 raplGAP was partially purified by precipitation using ammonium sulfate at 50% saturation. Protein was redissolved in a volume of phosphate-buffered saline equal to the original volume of the serum and then used at a dilution of 1:20,00Ofor Western blotting procedures. The anti-“Glu-Glu” is a mouse monoclonal antibody raised against a synthetic peptide derived from the sequence of polyoma middle T antigen (Grussenmyer et al., 1985). Cell Lines The derivations of the cell lines are as follows: SK-NEP-1 and G-401, human kidney, Wilms’tumor; 293, immortalized embryonal kidney cell; NIH 3T3, established mouse fibroblast; src-3T3, NIH 3T3 stably transformed byv-src (a gift of Steve Martin, University of California at Berkeley); ras 3T3, NIH 3T3 stably transformed by H-ras leu 61 mutant; HL60, human blood myeloblast, differentiation was with 1% dimethylsulfoxide and 1 Om5M dexamethasone for 5 days; MCF-7, human breast adenocarcinoma; MRC-5, human fetal lung; Genoa, hepatic adrenal carcinoma; Pant-1, human pancreatic carcinoma; SK-NS-H, human neuroblastoma; Wish, human amnion cell; Ovcar-4, human ovarian carcinoma; SK-UT-l, human uterus mesodermal tumor; KG-I, human blood myeloblast; IM-9, human blood lymphoblast; Scaber, human bladder carcinoma; and SK-MEL-3, human skin melanoma. Electrophoretic Procedures SDS-polyacrylamide gel electrophoresis was carried out essentially as described by Laemmli (1970) using 8% polyacrylamide gels. Western blotting was performed using a MiniProteantransblot apparatus

Cloning 1041

of raplGAP

(Bio-Rad Inc., Richmond, CA) according to the manufacturers instructions. mRNA was prepared from mammalian cell lines by the GITC method (Ullrich et al., 1977) or, for kidney, brain, and HeLa cells, it was purchased from Clonetech, Inc. Poly(A)t RNA (4 ug) was electrophoresed in a 1% agarose gel containing 2.2 M formaldehyde in 1 x MOPS bufferasdescribed byManiatisetal.(1982).Gelswerecapillaryblotted overnight onto nitrocellulose filters using 20 x SSC. Blots were probed with the radiolabeled 278 bp PCR fragment described in the section “Isolation of cDNA Clones.” Generation of PCR fragments from mRNA was performed using 50 ng poly(A)+ mRNA and the PCR kit according to the manufacturers instructions (Perkin-Elmer Cetus). Other Methods Competition binding analysis with raplGAP was performed essentially as described previously for rasGAP (Martin et al., 1990). raplGAP was used at a concentration of 15 nglml in a final volume of 30 ul. Protein assays were carried out using the method of Bradford (1978). Analysis of amino acid sequence homologies was carried out using the FASTA software program (Genetics Computing Company, Madison, WI) and the National Biomedical Research Foundation (NBRF) protein data base. Acknowledgments We thank Drs. Channing Der and Jean de Gunzburg for the gifts of recombinant rab and rap2 proteins, respectively. We are also indebted to Lauri Goda, Corey Levinson, and Dragan Spasic of the Cetus nucleic acid synthesis group and to Greg Eichinger of the Cetus sequencing facility. We thank Rob Halenbeck for providing the human tissue samples This work was partly funded by Hoffman-LaRoche and NCDDG grant #CA51992-02 (to F. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adverfisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

March

29, 1991.

proteins

in vesicular

transport.

Ballester, R., Marchuk, D., Boguski, M., Saulino, A., Letcher, R., Wigler, M., and Collins, F. (1990). The NF7 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins, Cell 63, 851-859. Bielinski, D. F., Morin, P. J., Dickey, 8. F., and Fine, R. E. (1989). Low molecular weight GTP-binding proteins are associated with neuronal organelles involved in rapid axonal transport and exocytosis. J. Biol. Chem. 264, 18383-18387. Bourne, H. R., Sanders, D.A., and McCormick, F. (1990). TheGTPase superfamily. A conserved switch for diverse cell functions, Nature 348, 125-132. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72, 248-252. Burstein, E. S., Linko-Stentz, K., Lu, Z., and Macara, I. G. (1991). Regulation of the GTPase activity of the ras-like protein ~25’~“~~. J. Biol. Chem. 266, 2889-2892. Campa, M. J., Chang, K.-G., Molina y Vedia, L., Reep, B. R., and Lapetina, E.G. (1991). Inhibition of ras-inducedgerminal vesicle breakdown in Xenopus oocytes by rap-l B. Biochem. Biophys. Res. Commun. 774, 1-5. Chardin, P., Boquet, P., Madaule, P., Popoff, M. R., Rubin, E. J., and Gill, D. M. (1989). The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinurn exoenzyme C3 and affects actin microfilaments in Vero cells. EMBO J. 8, 1087-1092. Chavrier, P., Parton, R. G., Hauri, H. P., Simons, K., and Zerial, (1990). Localization of low molecular weight GTP binding proteins exocytic and endocytic compartments. Cell 62, 317-329.

in platelets.

Biochem.

Biophys.

Res. Commun.

749,

Fischer, T. H., Gatling, M. N., Lacal, J.-C., and White, G. C. (1990). raplB, a CAMP-dependent protein kinase substrate, associates with the platelet cytoskeleton. J. Biol. Chem. 265, 19405-19408. Fischer von Mollard, G., Sudhof, GTP-binding protein dissociates cytosis. Nature 349, 79-81,

T. C., and Jahn, R. (1991). A small from synaptic vesicles during exo-

Frech, M., John, J., Pizon, V., Chardin, P., Tavitian, A., Clark, R., McCormick, F., and Wittinghofer, A. (1990). Inhibition of GTPase activating protein stimulation of Ras-p21 GTPase by the Krev-1 gene product. Science 249, 169-171. Garret, M. D., Self, A. J., van Oers, C., and Hall, A. (1989). Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins, J. Biol. Chem. 264, 10-13. Grussenmyer, T., Scheidtmann, K. H., Hutchinson, M. A., and Walter, G. (1985). Complexes of polyoma virus medium T antigen and cellular proteins. Proc. Natl. Acad. Sci. USA 82, 7952-7954. Hagag, N., Halegoua, S., and Viola, M. (1986). Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras ~21. Nature 379, 880-682. Halenbeck, R., Crosier, W. J., Clark, R., McCormick, F., and Koths, K. (1990). Purification, characterization and western blot analysis of human GTPase-activating protein from native and recombinant sources. J. Biol. Chem. 265, 21922-21928. Hall, A. (1990a). The cellular Science 249,635-640. Hall, A. (1990b). 923.

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ras and GAP-who’s

of small GTP-binding controlling

whom?

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Cell 67,921-

Hata, Y., Kikuchi, A., Sasaki, T., Schaber, M. D., Gibbs, J. B., and Takai, Y. (1990). Inhibition of the ras ~21 GTPase-activating proteinstimulated GTPase activity of c-Ha-ras p21 by smg p21 having the same putative effector domain as ras ~21s. J. Biol. Chem. 265,710471 07. Haubruck, H., Polakis, P., McCabe, P., Conroy, L., Clark, R., Innis, M. and McCormick, F. (1991). Mutational analysis of rapl/Krev protein; sensitivity to GTPase activating proteins and suppression of the yeast cdc24 budding defect. J. Cell. Biochem. [Suppl] 758, 138.

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Kaplan, D. R., Morrison, D. K., Wong, G., McCormick, F., and Williams, L. T. (1990). PDGF 8-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell 67, 125133. Kawata, M., Kikuchi, A., Hoshijima, M., Yamamoto, K., Hashimoto, E., Yamamura, H., and Takai, Y. (1989). Phosphorylation of smgp21, a ras pal-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin E in intact human platelets. J. Biol. Chem. 264, 15688-15695. Kazlauskas, A., Ellis, C., Pawson, T., and Cooper, J. A. (1990). Binding of GAP to activated PDGF receptors. Science 247, 1578-1581. Kikuchi, A., Sasaki, T., Araki, S., Hata, Y., and Takai, Y. (1989). Purification and characterization from bovine brain cytosol of two GTPaseactivating proteins specific for smg ~21, a GTP-binding protein having the same effector domain as c-ras ~21s. J. Biol. Chem. 264, 91339136. Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y., and Noda, M. (1989). Aras-related gene with transformation suppressor activity. Cell 56, 77-84. Kitayama, H., Matsuzaki, T., Ikawa, Y., and Noda, M. (1990). Genetic analysis of the Kirsten-ras-revertant 1 gene: potentiation of its tumor suppressor activity by specific point mutations. Proc. Natl. Acad. Sci. USA 87, 4284-4288. Korn, L. J., Siebel, C. W., McCormick, F., and Roth, R. A. (1987). Ras p21 as a potential mediator of insulin action in Xenopus oocytes. Science 236, 840-843. Kozak, M. (1984). Point mutations close to the AUG affect the efficiency of translation of rat preproinsulin 308, 241-246.

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Miaorella, B., Inlow, D., Shauger, A., and Harano, D. (1988). Largescale insect cell culture for recombinant protein production. Bio/Technology 6, 1508-1510. Mulcahy, L. S., Smith, M. R., and Stacey, D. W. (1985). Requirement for ras proto-oncogene function during serum stimulated growth of NIH-3T3 cells. Nature 373, 241-243. Munemitsu, S., Innis, M., Clark, R., McCormick, F., Ullrich, A., and Polakis, P. (1990). Molecular cloning and expression of G25K cDNA, the human homolog of the yeast cell cycle gene cdc42. Mol. Cell. Biol. 10, 59774982. Nagata, K.-l., Nagao, S., and Nozawa, Y. (1989). Low M,GTP-binding proteins in human platelets: cyclic AMP-dependent protein kinase phosphorylates m22KG(l) in membrane but not c21KG in cytosol. Biochem. Biophys. Res. Commun. 180, 235-242. Pizon, V., Lerosey, I., Chardin, P., andTavitian. A. (1988a). Nucleotide sequence of a human cDNA encoding a ras-related protein (raplB). Nucl. Acids Res. 16, 7719. Pizon, V., Chardin, P.. Lerosey, I., Olofsson, B., and Tavitian, A. (1988b). Human cDNAs rap1 and rap2 homologous to the Drosophila gene Dras3 encode proteins closely related to ras in the ‘effector’ region. Oncogene 3, 201-204. Polakis, P. G., Weber, R. F., Nevins, B., Didsbury, J. R., Evans, T., and Snyderman, R. (1989). Identification of the raland racl gene products, low molecular mass GTP-binding proteins from human platelets. J. Biol. Chem. 264, 16383-16389. Polakis, P. G., Rubinfeld, B., Evans, T., and McCormick, F. (1991). Purification of plasma membrane-associated GTPase-activating protein specific for rapl/Krev-I from HL60 cells. Proc. Natl. Acad. Sci. USA 88, 239-243. Quinn, M. T., Parkos, C. A., Walker, L., Orkin, S. H., Dinauer, M. C., and Jesaitis, A. J. (1989). Association of a Ras-related protein with cytochrome b of human neutrophils. Nature 342, 198-200. Sanders, D. A. (1990). A guide to low molecular Growth Diff. 1, 251-258. Sanger, F., Nicklen, with chain-terminating 5467.

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Cell

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Smith, G. E., Summers, M. D., and Fraser, human B-interferon in insect cells infected sion vector. Mol. Cell. Biol. 3, 2156-2165.

M. J. (1983). Production of with a baculovirus expres-

Summers, M. D., and Smith, G. E. (1987). A Manual of Methods Baculovirus Vectors and Insect Cell Culture Procedures. Texas ricultural Experiment Station Bulletin no. 1555.

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Touchot, N., Chardin, P., and Tavitian, A. (1987). Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library. Proc. Natl. Acad. Sci. USA 84, 8210-8214. Trahey, M., Wong, G., Halenbeck, R., Rubinfeld, B., Martin, G. A., Ladner, M., Long, C., Crosier, W. J., Watt, K., Koths, K., and McCormick, F. (1988). Molecular cloning of two types of GAP complementary DNA from human placenta. Science 242, 1897-1700.

Ullrich, A., Shine, J., Chirgwin, J., Pictet, R., Tischer, E., Rutter, W. J., and Goodman, H. M. (1977). Rat insulin genes: construction of plasmids containing the coding sequences. Science 796, 1313-1316. Vogel, U. S., Dixon, R. A. F., Schaber, M. D., Diehl, R. E., Marshall, M. S., Scolnick, E. M., Sigal, I. S., and Gibbs, J. B. (1988). Cloning of bovine GAP and its interaction with oncogenic ras ~21. Nature 335, 90-93. Waldo, G. L., Evans, T., Fraser, E. D., Northup, J. K., Martin, M. W., and Harden, T. K. (1987). Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go. Biochem. J. 246,431-439. Xu, G., Lin, B., Tanaka, K., Dunn, D., Wood, D., Gesteland, R., White, R., Weiss, R., and Tamanoi, F. (1990). The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae. Cell 63, 835-841. GenBank

Accession

The accession M64788.

number

Number for the sequence

reported

in this article

is