Regulation of p21ras activity

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T h e ras genes participate in normal cell growth and are mutationally activated in many different types of tumor 1. The involvement of ras in these physiological and pathological processes has generated considerable interest in characterizing its normal functions, understanding how these functions are regulated, and elucidating the mechanisms by which ras contributes to tumorigenesis. The ras genes belong to a superfamily of genes encoding proteins that bind guanine nucleotides GDP and GTP with high affinity and possess intrinsic GTPase activity2-5. The Ras proteins function as molecular switches that are active when GTP is bound to them and inactive in the GDP-bound form (Fig. 1). The activity of Ras proteins is highly regulated, so under most growth conditions only a small percentage is in the active, GTP-bound form. By contrast, murationally activated r a s genes encode proteins whose proportion of GTP-bound molecules is much higher than that of normal Ras proteins. The location of Ras proteins at the plasma membrane, combined with their growth-promoting activity, have made them prime candidates for transducers that help mediate mitogenic signals from growth factor receptors to downstream targets. This review emphasizes our evolving notions of the control mechanisms that normally regulate the biological activity of mammalian Ras proteins, and the perturbation of this regulation in tumors. Although it is mitogenic in many situations, ras induces differentiation in some experimental systems, including neurite formation in the rat PC12 pheochromocytoma cell line 6 and adipocyte differentiation of 3T3-L1 fibroblasts 7. Whether ras induces growth or differentiation is determined by the cell; in both situations, signaling through ras involves similar mechanisms ~. The relative biological activity of a given mutant ras gene is similar whether growth or differentiation is being measured, which suggests that Ras proteins are functioning in an analogous manner regardless of the phenotype that they induce.

Functional organization of Ras proteins Most eukaryotes, including S a c c h a r o m ~ c e s cerev i s i a e 9 and mammals 1, contain more than one ras gene. Mammals have three well-defined ras genes H a - r a s , K i - r a s and N - r a s - one or more of which are expressed in virtually all tissues 1°,11. The yeast and mammalian Ras proteins are sufficiently conserved that they can be biologically active in the heterologous system. Each mammalian r a s gene encodes a 189 amino acid primary translation product derived from four coding exons. The proteins migrate in gels as 21 kDa proteins, which has ted to their being called p21 or p21 ras. There are actually four mammalian Ras proteins because Ki-ras, which contains two different exons number 4, undergoes differential splicing, producing

Nomenclature: ra~ Gene, generic use. RAS: Yeast ras gene. Ras: Protein, generic use. RAS: Yeast Ras protein. IRA1 and IRA2 (and NF1) follow rules for yeast RAS/RAS.

Regulation of p21 ras activity DOUGLAS R. LOWY~KE ZHANG,JEFFREYE. DECLUE AND BERTHEM. WILLUMSEN

The ras genes encode GTP/GDP-bindingproteins that participate in mediating mitogenic signals from membrane tyrosine kinases to downstream targets. The activity of p21 gasis determined by the concentration of GTP-p21TM, which is tightly regulated by a complex array of positive and negative control mechanisms. GAP and NFI can negatively regulate p21 m activity by stimulating hydrolysis of GTP bound to p21 r~. Other ceUularfactors can positively regulate 1)21ras by stimulating GDP/GTP exchange. two p21 proteins that differ according to which of the two exons (4A or 4B) encodes the 25 carboxy-terminal amino acids. Mutagenesis of ras genes and X-ray crystallographic analysis of Ras proteins have provided considerable information regarding the structure of p21 and its functional organization. The Ras proteins can be divided into three regions: a CAAX box, a heterogeneous region and a catalytic domain (Fig. 2). The carboxy terminus is primarily responsible for attachment of the protein to the membrane, which is required for biological activity12. Ras proteins are cytosolic when synthesized, and they become attached to the membrane after several post-translational modifications at the carboxy terminus. The last four amino acids of the primary translation products form the socalled CAAX box, a motif subject to several post-translational modifications, which is common to the carboxy terminus of many different classes of protein 13. C is an invariant cysteine common to all Ras proteins, A tends to be an aliphatic (nonpolar) amino acid, and X can be one of several amino acids. Post-translational processing involves farnesylation of the cysteine, cleavage of the three carboxy-terminal amino acids, and carboxymethylation of the cysteine (reviewed in Ref. 14). These modifications, together with palmitoylation of cysteines located in the heterogeneous region immediately upstream of the famesylated cysteine 15, serve to anchor the protein firmly to the plasma membrane. The 20 amino acids of the heterogeneous region are almost completely divergent among the four p21 proteins. For each protein, however, the amino acid sequence of the heterogeneous region is highly conserved between humans and rodents. The heterogeneous region encoded by exon 4B of K i - r a s does not contain any cysteines; in this protein, a run of six lysine residues within this region increases the affinity of the protein for the membrane, as do the palmitoylated cysteines in the other p21 proteins 15. The number and location of the palmitoytated cysteines are distinct in each of the other three p21 proteins. These differences in the secondary membrane association signal of the Ras proteins might confer different activities on the p21 variants.

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The activation/inactivation cycle of p21 ra-~and its regulation by cellular factors. Input signals are transmitted through activated growth factor receptors, resulting in the activation of p21 by stimulating the release of GDP (exchange factors), inhibiting GTPase stimulators (GAP, NF1), or both. The binding of GTP to p21 allows the molecule to present its effector domain (black rectangle) in a conformation that can be recognized by both GTPase-stimulating proteins (GAP, NF1) and target effector molecule(s). The hydrolysis of GTP to GDP returns p21 to the inactive state. See text for a discussion of the potential role of GAP and NF1 as targets for p21. The amino-terminal 165 amino acids represent the Point mutations that activate the biological activity catalytic domain. Overall this region is more than 90% of p21 invariably map to the catalytic domain. The homologous among the three mammalian ras genes, mutants fall into two main classes. Some mutants, with the amino-terminal 86 amino acids being identfound most often in tumors, encode proteins that are ical. X-ray crystallography of the amino-terminal 171 resistant to regulation by the GTPase-accelerating amino acids shows that this domain is composed of a activity of GAP and NF1 proteins (see below) and thus central core of two ~-sheets surrounded by a peripherremain bound to GTP for a relatively long period 1. ally located series of 0t-helices and loops 16,17. The These include mutations of codons 12, 13, 59 and 61. guanine nucleotide binds in a pocket formed by Mutants in the other class encode proteins showing an several segments of the catalytic domain. Mutation of increased rate of nucleotide dissociation. This results any of these segments can drastically reduce nucleoin a higher level of GTP binding in vivo because tide binding. release of GDP is rate limiting for the normal protein. The GTPase activity of the catalytic domain and its Mutation of codons 116, 119 and 146 can result in nucleotide dissociation rate control the activation mutants with this phenoWpe. state of the protein. The high GTP:GDP ratio in cells means Catalytic Heterogeneous CAAX that GTP binds to any nucleotide-free p21 formed by dissociation, since the affinity ] Effector n6 ]46 of Ras proteins for GTP is of I 1 I I the same order of magnitude I V//A -• as that for GDP. The GTP-p21 protein hydrolyses GTP to rlTmTrrl [] GDP at a low rate, converting GAP binding the Ras protein from active binding GTP-p21 to inactive GDP-p21. As discussed below, both GTP FIG~i hydrolysis and nucleotide reLinear representation of the structural and functional domains of p21 ras. Amino terminus is lease are stimulated by cellular shown at the left. while the carboxy terminus is on the right. Regions required for effector components in a highly regufunction, GAP interaction, and membrane binding are indicated. Black dots (°) represent lated manner. amino acid residues at which missense mutations enhance the transforming potential of p21.

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TI(3 NOVEMBEK/DECEMBER1991 VOL. 7 NO. 11/12

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[~EVIEWS The greater biological activity of mutant p21 seems to represent a quantitative difference from normal p21, rather than a qualitative change. As is true of normal p21, mutant p21 is inactive when bound to GDP. Conversely, when normal p21 is bound to a nonhydrolysable GTP analog, its biological activity closely resembles that of p21 carrying a mutation at residue 12 or 61 (Refs 18, 19). The catalytic region of the protein also contains a domain implicated in p21-target interaction. This effector region was defined genetically by mutants that were inactive in mammalian cells and yeast although they encoded stable proteins that localized to the membrane and could still bind guanine nucleotides20.2L Linker scanning mutants involving codons 22-43 possessed these phenotypic characteristics, and point mutational analysis of codons 32-40 showed that such mutants exhibited the same phenotype as the linker scanning mutants. Recent analysis of chimeras formed between ras and a ras-related gene, rap-la, which can antagonize ras-induced transformation, has identified the p21 rasspecific amino acids that can convert p21rap to a transforming gene 22. In these two genes, codons 32-44 encode the same amino acids. While ras codons 20-31 or 45-54 separately are insufficient to make a chimeric gene transforming, the combination of ras codons 20-31 and 45-54 specifies a transforming gene. These results indicate that the p21 ras effector region involves at least some amino acids amino-terminal to residue 32 and carboxy-terminal to residue 44. All of residues 23-55, although forming parts of different structures in the protein, are on a solvent-exposed surface of the catalytic domain, where they might be accessible for interaction with other macromolecules. The conformation of parts of this segment has been shown to change depending upon whether GTP or GDP is bound to the protein ~(',lv.

Ras regulation The half-life of p21 is greater than 24 hours, suggesting that much of its regulation is at the level of protein activity. Theoretically, p21 activity could be regulated by modulating either the proportion of GTPbound p21 or the membrane association of the protein. There is as yet no evidence that various growth states are associated with changes in the affinity of the protein for the membrane, although the palmitate on p21 does have a short half-life (about 20 minutes) 2~. Attention has therefore been focused upon regulation of p21 activity by the concentration of GTP-p21.

Negative regulation: GAP and NF1 Injection of GTP-p21 into cells has demonstrated that hydrolysis to GDP-p21 occurs much more rapidly in vivo than in vitro, suggesting that GTP hydrolysis may be enzymatically regulated in cells. This observation led to the identification of GAP (GTPase-activating protein), a 120 kDa cytoplasmic protein that can accelerate the intrinsic GTPase activity of GTP-p21 ras by more than two orders of magnitude (Fig. 1) 19. The normal proteins encoded by all three mammalian ras genes are fully sensitive to GAP, as is the closely related p21 ~-ra-"(Refs 24-26). However, GAP fails to accelerate

the intrinsic GTPase activity of the GTP-bound form of several other members of the supeffamily of Ras-related proteins 26,27. Most significantly, Ras proteins activated by mutations at residues 12 or 61 are resistant to the GTPase-accelerating activity of GAP 19. Thus the difference in biological activity seen in many biological systems between activated Ras and normal Ras could be explained by resistance of the activated proteins to downregulation by GAP. The G A P gene appears to be expressed as widely as ras in mammalian tissues. The gene is conserved in evolution, and GAP protein isolated from nonmammalian species is active on mammalian p21 ras in vitro 24. Although GAP activity on mammalian p21 ra-' has not been identified in S. cerevisiae, yeast do contain at least t w o GAP-related genes zS, IRA1 and IRA2. The proteins encoded by I R A I and I R A 2 share homology with the carboxy-terminal one-third of GAP, which is the domain of GAP that contains the catalytic activity (Fig. 3) 29. The IRA1 and IRA2 proteins negatively regulate yeast RAS proteins, apparently via a GTPase acceleration mechanism similar to that of mammalian GAP. However, they are not enzymatically active against mammalian p21 ra~ (Ref. 30), which correlates with the observation that in S. cerevisiae nonmutated mammalian ras behaves as a partially activated ras gene31. Direct evidence that GAP can negatively regulate Ras in vivo has come from other studies in yeast and mammalian cells. Although the IRA genes are inactive against mammalian ras, mammalian G A P can substitute functionally for IRA1 in yeast, since yeast RAS protein is sensitive to negative regulation by GAP 28,32, Furthermore, overexpression of GAP in NIH 3T3 cells can prevent and reverse transformation induced by overexpression of normal ras, but not of mutant r a s 33. G A P overexpression can also negatively regulate cell transformation by src, an oncogene whose biological activity in NIH 3T3 cells is known to require endogenous p21 (Refs 34, 35). The portion of GAP that encodes only the catalytic domain was also active in suppressing the transforming activity of src and normal ras, but not mutant ras. Taken together, the results strongly suggest that ras is negatively regulated via the catalytic activity of GAP. When the sites of interaction between p21 and GAP were analysed, GAP was found to bind specifically to the p21 effector region. The interaction with GAP depended upon the binding of GTP to p21, and all biologically active p21 proteins tested bound GAP 24,25,29. As discussed below, these observations suggested that GAP might serve as a downstream target of p21, in addition to being an upstream negative regulator. GAP also appears to interact with a small loop that includes residue 61, which is hypothesized to be critical for initiating GTP hydrolysis 16. Analysis of p21 molecules encoded by chimeric ras-rap genes has shown that p21 r'~ residues 61-65 are sufficient to make p21rap (which binds to GAP but is normally resistant to its GTPase-accelerating activity) sensitive to the catalytic activity of GAP~,. Since the loop formed by amino acids 59-65 assumes various conformations, only one of which seems to permit efficient GTP hydrolysis ~6, these results suggest that the catalytic

"FIGNOVEMP,ER/DECEMBER1991 VOL.7 ~O. 11/12

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[~EVIEWS

activity of GAP may involve SH2 stabilization of the loop in this / % permissive conformation. GAP The NF1 gene responsible for von Recklinghausen neuroI CATALYTIC SH3 fibromatosis encodes a protein with properties similar to GAP. NF1 [ A portion of the NF1 protein is homologous to the catalytic domain of GAP, and a larger region is also homologous to IRA1 [ the IRA proteins (Fig. 3) 37. Functional studies have thus far been limited mainly to IRA2 [ analysis of the GAP-related domain of NF1 (Refs 37-39). HGE This region of the protein displays a GTPase-accelerating Domain structure of GAP and proteins that contain a GAP homology domain. Amino activity that is similar to that of terminus is shown at the left, while the carboxy terminus is on the right. The src homology regions 2 and 3 (SH2 and SH3), which lie at the amino terminus of GAP, mediate the GAP, in that it is active on the association of GAP with protein tyrosine kinases and other cellular proteins. The normal versions of the procarboxy-terminal 350 amino acids of GAP (black rectangle) are necessary and sufficient for teins encoded by the three GTPase stimulation of p21 ~. The type 1 neurofibromatosis (NF1) protein and the products mammalian ras genes, and of the yeast genes IRA1 and IRA2 contain a similar GAP-related domain, but lack SH2 and inactive both on several other SH3 regions. A large additional region of homology between NF1 and the IRA genes is members of the superfamily of shown (hatched region). Ras-related proteins and on Ras proteins that have been mutationally activated at defective for CDC25. Analysis of this cDNA revealed residue 12 or 61. As was true of GAP, the GAP-related an open reading frame of almost 300 amino acids that domain of NF1 can substitute functionally for IRAI in is about 35% homologous to the catalytic region of yeast. These observations suggest that NF1 may be yeast CDC25 (E. Sturani, pers. commun.). another negative regulator of mammalian ras (Fig. 1). Further support for this hypothesis comes from the Signal u'ansductionby"Sas Ras has been shown to participate in serum- and finding that a high proportion of p21 ras was in a GTPbound form in a Schwannoma cell line, derived from a growth factor-induced mitogenesis in primary and patient with NF1 neurofibromatosis; the cell line con- established cells 46. Microinjection of a Ras-specific tains normal levels of GAP but lacks detectable NF1 monoclonal antibody into cells can inhibit various mitotic stimuli, including those induced by serum, protein (J.E. DeClue and D.R. Lowy, unpublished). platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), a constitutively active CSF-1 Positive regulation:stimulatedGDP/GTPexchange At least under some conditions, the guanine nu- receptor (v-fins gene producO, two non-receptor tyrocleotide appears to dissociate from p21 significantly sine protein kinases (v-src and v-fes gene products), or faster in vivo than in vitro 4°. There have been several a mutationally activated p21. The specificity of the reports of activities in cells that stimulate nucleotide neutralizing antibody in vivo has been validated by exchange in vitro, but the relevance of these activities showing that it does not inhibit mitogenesis induced by two constitutively active serine/threonine kinases to the regulation of p21 in vivo remains to be demonstrated. One group has purified a 35 kDa membrane- ( v - m o s and v - r a f g e n e products) or by a biologically associated protein that appears to be conserved active mutant p21 that did not bind the monoclonal among vertebrates 41. In addition to p21 ras, this factor antibody. Furthermore, a dominant inhibitory r a f stimulates guanine nucleotide release from other prod- mutant will prevent transmission of the ras mitogenic ucts of the ras-related superfamily. Two groups have signaPL These observations place ras downstream isolated a cytoplasmic activity that also stimulates from receptor and non-receptor tyrosine protein kinases guanine nucleotide exchange 42,43. The molecular mass and upstream from raf. In intact cells, an increase in the concentration of of these activities was substantially higher than 35 kDa, which suggests they may be distinct from the GTP-p21 can be detected within minutes of ligandstimulated activation of tyrosine kinases 48,49. In T cells, membrane-associated protein. In S. cerevisiae, guanine nucleotide exchange on which apparently have a very high constitutive nucleotide exchange rate on p21, the increase in yeast RAS protein appears to be stimulated by CDC25, a membrane-associated, upstream positive regulator of GTP-p21 after phorbol ester-induced protein kinase C yeast RAS44. The catalytic domain of a related yeast activation has been reported to be associated with a protein, SCD25, can stimulate guanine nucleotide ex- decrease in GAP-like activity4°. The mechanism leading to increased GTP-p21 in change on yeast and mammalian Ras protein in vitro and in vivo "~5. The relevance of these observations to other cell types is unclear. Several potentially importmammalian ras regulation is suggested by the ability ant changes in GAP have been reported to occur in of a mouse cDNA to complement yeast cells that are fibroblasts shortly after activation of the PDGF receptor. TIG NO'v~MBER/DECEMBER1991 VOL.7 .NO. 11/12

m

IB!EVlEW These alterations include phosphorylation of GAP on tyrosine, an increase in the affinity of tyrosine-phosphorylated GAP for the membrane, and the physical association of GAP with the activated PDGF receptors, with a membrane-associated 62 kDa protein, and with a cytoplasmic 190 kDa protein 50,51. The association between GAP and these proteins requires the SH2 (and possibly the SH3) motifs located in the amino terminus of GAP (Fig. 3). Serum induces production of lipids that can inhibit the catalytic activity of GAP 52, but its physiological significance is still under investigation 53. No changes have been reported in the GTPase-accelerating activity of membrane-associated GAP, but a fourfold reduction in catalytic activity has been noted for GAP associated with the 190 kDa protein 51. The relevance of these changes to the increased concentration of GTP-p21 is unclear, since activation of other receptors, such as those for CSF-1 and insulin, increases the concentration of GTP-p21 without tyrosine phosphorylation of GAP or complex formation. Identification of the immediate downstream target(s) with which mammalian ras interacts to transmit its signal remains one of the major unresolved questions of ras biology. Adenylyl cyclase is a major target of RAS in yeast cells but not in mammalian cells31. By analogy with yeast R_AS, however, it is reasonable to believe that p21 r'~ functions in mammalian cells by regulating one or more catalytic proteins. As noted above, genetic and biochemical analysis has shown that the interaction between p21 and GAP is consistent with the idea that GAP is a downstream ras target 24,2s,29. Less extensive characterization has been carried out for NF1, but its catalytic function also appears to require binding to the p21 effector region, which also makes it a potential downstream target. There are precedents among other guanine nucleotidebinding proteins for bifunctional regulation by a single molecule 4. As reviewed recently in T/G 54, studies carried out thus far do not permit an unequivocal conclusion regarding the potential p21 target function of GAP and NF1. If GAP does represent a downstream target of p21, its tyrosine kinaseqnduced translocation to the membrane, where p21 is active, and its association with membrane proteins might be viewed as a mechanism for increasing the concentration of target molecules. In one system, p21 and GAP function together to inhibit the closing of ion channels in isolated membranes 55. Furthermore, the binding of the p21 effector region to GAP mimics important aspects of the presumed interaction between p21 and its downstream target(s). For example, it is thought that p21raP antagonizes p21ras-induced transformation by competing for the p21 r~ target, and in vitro competition studies 56 have shown that the p21rap binds to GAP more tightly than does p21 ra~. On the other hand, p21 ras carrying an effector domain that is temperature sensitive for transformation is equally sensitive to the catalytic activities of GAP and NF1 at permissive and nonpermissive temperatures 57. These results suggest at least that the negative regulatory function of GAP and its hypothetical target function can be dissociated. In summary, ras plays a critical role in signal transduction. Control of p21 activity by GAP represents the

first clear example of a direct, physiologically significant interaction between p21 and another protein. Since regulation of ras is likely to be complex, additional molecules that interact with p21 will probably be identified, and their activities in rum may be highly regulated. Elucidation of these controls and the pathway through which ras functions should bring important insights into normal and abnormal cell growth..

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

39 40

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41 Huang, Y.K., Kung, H.F. and Kamata, T. (1990) Proc. Natl Acad. Sci. USA 87, 8008-8012 42 Wolfman, A. and Macara, I.G. (1990) Science 248, 67--69

4.3 Downward, J., Riehl, R., Wu, L. and Weinberg, R.A. (1990) Proc. Natl Acad. Sci. USA 87, 5998-6002 44 Jones, s., Vignais, M.L. and Broach, J.R. (1991) Mol. Cell. Biol. 11, 2641-2646 45 Rey, I. et al. (1991) Oncogene6, 347-349 46 Smith, M.R., DeGudicibus, S.J. and Stacey, D.W. (1986) Nature 320, 540-543 47 Kolch, W.. Heidecker, G., Lloyd, P. and Rapp, U.R (1991) Nature 3"49, 426--428 48 Satoh, T. et aL (1990) Proc. Natl Acad. Sci. USA 87, 5993-5997 49 Gibbs, J.B. et aL (!1990)"J. Biol. Chem. 265, 20437-20442 50 Molloy, C0. etal. (1989) Nature342, 711-714

D . R Lowr,, K ZHANG AND J,E. I)ECLUE ARE IN THE LABORATORY OF CELLULAR ONCOLOGY, NATIONAL CANCER INSTITUTE, BETHESDA, M D 20892, USA," R M . WILLUMSEN IS IN THE UNIVERSITY INSTITUTE OF MICROBIOLOGY, D K 1353 COPENHAGEN K, DENMARK.

T h e non-receptor or cytoplasmic tyrosine kinases are encoded by the abl, arg and Jps genes, and by the nine src-related genes src, yes, fyn, fgr, lck, lyn, bck, blk and tkl (Ref. 1). The role of these molecules in normal signal transduction is not well characterized, although recent studies on lck and f y n indicate that their products may be involved in the response of T cells to antigen 2,3. The discovery of non-receptor tyrosine kinase homologs in Drosophila - there are currently four known Drosophila homologs, abl, Dsrc29A, Dsrc64B and fps (Refs 4-6) - permits the application of genetic strategies to the question of what kinds of developmental events are regulated by non-receptor tyrosine kinase-mediated signal transduction. Recently, gene disruption has been used to ask this question for the mouse c-abl and c-src genes 7-0. The surprising result from the studies on Drosophila abl and murine c-src and c-abl is that only subtle phenotypes result from mutations in these genes. To understand this result better, we have exploited Drosophila genetics to screen for second-site modifiers of the abl mutant phenotype. The Drosophila abl gene was isolated by DNA cross-hybridization with the murine v-abl oncogene 4,1°. The deduced amino acid sequence is more similar to the murine and human c-Abl and c-Arg proteins than to any other known tyrosine kinase in vertebrates or Drosophila TM. The Drosophila Abl protein is most similar to mammalian c-AN in the catalytic domain and in the regulatory domains SH2 and SH3 (Fig. 1). The carboxy-terminal domain of the Drosophila

1

F, MICHAELHOFFMANN

Genetic studies on Drosophila Abl and, more recently, on mouse c-Abl and c-Src indicate that the functions of these non-receptor tyrosine kinases may duplicate activities of other molecules within signal transduction pathways. In Drosophila, second-site mutations have been recovered that disrupt the redundant functions so that the Abl tyrosine kinase is essential to the formation of axonal connections in the embryonic central nervous system and for attachment of embryonic muscles to the body wall Molecular isolation and analysis of the genes identified by these second-site mutations should define the molecular basis for the genetic redundancy. protein has weak but statistically significant sequence similarity with the analogous region of tile mouse c-Abl protein. The abl gene is expressed in a broad variety of tissues and times during Drosophila development. Its mRNA is initially supplied to the early embryo maternally. The first zygotic synthesis of abl mRNA and protein is detected midway through cmbuogenesis. The zygotic mRNA and protein are most abundant in the

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Domains of the Drosophila Abl protein and their amino acid sequence similarities and identities to routine c-AN (tspe lb) a> determined by the University of Wisconsin Genetics Computer Group Bestfit program. TIG NOVf:,MBER/1)FCEN[BER1991 VOL. 7 NO. 11/12 g D)Ol Flst'xicl Scicn~v' Puhlisht,rs l i d ~1 K (1168 0179 91 $O2 I)~l

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