The extreme carboxyl terminus of v-Abl is required for lymphoid cell transformation by Abelson virus

The v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) induces transformation of pre-B cells in vivo and in vitro and can transform immortalized fibroblast cell lines in vitro. Although the kinase activity of the protein is required for these events, most previously studied mutants encoding truncated v-Abl proteins that lack the extreme carboxyl terminus retain high transforming capacity in NIH 3T3 cells but transform lymphocytes poorly. To understand the mechanisms responsible for poor lymphoid transformation, mutants expressing a v-Abl protein lacking portions of the COOH terminus were compared for their ability to transform pre-B cells. Although all mutants lacking sequences within the COOH terminus were compromised for lymphoid transformation, loss of amino acids in the central region of the COOH terminus, including those implicated in JAK interaction and DNA binding, decreased transformation twofold or less. In contrast, loss of the extreme COOH terminus rendered the protein unstable and led to rapid proteosome-mediated degradation, a feature that was more prominent when the protein was expressed in Ab-MLV-transformed lymphoid cells. These data indicate that the central portion of the COOH terminus is not essential for lymphoid transformation and reveal that one important function of the COOH terminus is to stabilize the v-Abl protein in lymphoid cells.     

The carboxyl terminus of v-Abl protein can augment SH2 domain function

Abelson murine leukemia virus (Ab-MLV) transforms NIH 3T3 and pre-B cells via expression of the v-Abl tyrosine kinase. Although the enzymatic activity of this molecule is absolutely required for transformation, other regions of the protein are also important for this response. Among these are the SH2 domain, involved in phosphotyrosine-dependent protein-protein interactions, and the long carboxyl terminus, which plays an important role in transformation of hematopoietic cells. Important signals are sent from each of these regions, and transformation is most likely orchestrated by the concerted action of these different parts of the protein. To explore this idea, we compared the ability of the v-Src SH2 domain to substitute for that of v-Abl in the full-length P120 v-Abl protein and in P70 v-Abl, a protein that lacks the carboxyl terminus characteristic of Abl family members. Ab-MLV strains expressing P70/S2 failed to transform NIH 3T3 cells and demonstrated a greatly reduced capacity to mediate signaling events associated with the Ras-dependent mitogen-activated protein (MAP) kinase pathway. In contrast, Ab-MLV strains expressing P120/S2 were indistinguishable from P120 with respect to these features. Analyses of additional mutants demonstrated that the last 162 amino acids of the carboxyl terminus were sufficient to restore transformation. These data demonstrate that an SH2 domain with v-Abl substrate specificity is required for NIH 3T3 transformation in the absence of the carboxyl terminus and suggest that cooperativity between the extreme carboxyl terminus and the SH2 domain facilitates the transmission of transforming signals via the MAP kinase pathway.     

Absence of p53 complements defects in Abelson murine leukemia virus signaling

The v-Abl protein encoded by Abelson murine leukemia virus (Ab-MLV) induces transformation of pre-B cells via a two-stage process. An initial proliferative phase during which cells with limited tumorigenic potential expand is followed by a crisis period marked by high levels of apoptosis and erratic growth. Transformants that survive this phase emerge as fully malignant cells and usually contain mutations that disable the p53 tumor suppressor pathway. Consistent with the importance of p53 in this process, pre-B cells from p53 null animals bypass crisis. Thus, the transformation process reflects a balance between signals from the v-Abl protein that drive transformation and those coming from the cellular response to inappropriate growth. One prediction of this hypothesis is that Ab-MLV mutants that are compromised in their ability to transform cells may be less equipped to overcome the effects of p53. To test this idea, we examined the ability of the P120/R273K mutant to transform pre-B cells from wild-type, p53 null, and Ink4a/Arf null mice. The SH2 domain of the v-Abl protein encoded by this mutant contains a substitution that affects the phosphotyrosine-binding pocket, and this mutant is compromised in its ability to transform NIH 3T3 and pre-B cells, especially at 39.5 degrees C. Our data reveal that loss of p53 or Ink4a/Arf locus products complements the transforming defect of the P120/R273K mutant, but it does not completely restore wild-type function. These results indicate that one important transforming function of v-Abl proteins is overcoming the effects of a functional p53 pathway.     

SH2-containing inositol 5'-phosphatase inhibits transformation of Abelson murine leukemia virus

v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) transforms pre-B cells. Transformation requires the phosphatidylinositol 3-kinase (PI3K) pathway. This pathway is antagonized by SH2-containing inositol 5'-phosphatase (SHIP), raising the possibility that v-Abl modulates PI3K signaling through SHIP. Consistent with this, we show that v-Abl expression reduces levels of full-length p145 SHIP in a v-Abl kinase activity-dependent fashion. This event requires signals from the Abl SH2 domain but not the carboxyl terminus. Forced expression of full-length SHIP significantly reduces Ab-MLV pre-B-cell transformation. Therefore, reduction of SHIP protein by v-Abl is a critical component in Ab-MLV transformation.     

Temperature-sensitive transformation by an Abelson virus mutant encoding an altered SH2 domain

Abelson murine leukemia virus (Ab-MLV) encodes the v-Abl protein tyrosine kinase and induces transformation of immortalized fibroblast lines and pre-B cells. Temperature-sensitive mutations affecting the kinase domain of the protein have demonstrated that the kinase activity is absolutely required for transformation. Despite this requirement, mutations affecting other regions of v-Abl modulate transformation activity. The SH2 domain and the highly conserved FLVRES motif within it form a phosphotyrosine-binding pocket that is required for interactions between the kinase and cellular substrates. To understand the impact of SH2 alterations on Ab-MLV-mediated transformation, we studied the Ab-MLV mutant P120/R273K. This mutant encodes a v-Abl protein in which the beta B5 arginine at the base of the phosphotyrosine-binding pocket has been replaced by a lysine. Unexpectedly, infection of NIH 3T3 or pre-B cells with P120/R273K revealed a temperature-dependent transformation phenotype. At 34 degrees C, P120/R273K transformed about 10-fold fewer cells than wild-type virus of equivalent titer; at 39.5 degrees C, 300-fold fewer NIH 3T3 cells were transformed and pre-B cells were refractory to transformation. Temperature-dependent transformation was accompanied by decreased phosphorylation of Shc, a protein that interacts with the v-Abl SH2 and links the protein to Ras, and decreased induction of c-Myc expression. These data suggest that alteration of the FLVRES pocket affects the ability of v-Abl to interact with at least some of its substrates in a temperature-dependent fashion and identify a novel type of temperature-sensitive Abelson virus.     

Disruption of the Shc/Grb2 complex during abelson virus transformation affects proliferation, but not apoptosis

The v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) induces pre-B-cell transformation. Signals emanating from the SH2 domain of the protein are required for transformation, and several proteins bind this region of v-Abl. One such protein is the adaptor molecule Shc, a protein that complexes with Grb2/Sos and facilitates Ras activation, an event associated with Ab-MLV transformation. To test the role this interaction plays in growth and survival of infected pre-B cells, dominant-negative (DN) Shc proteins were coexpressed with v-Abl and transformation was examined. Expression of DN Shc reduced Ab-MLV pre-B-cell transformation and decreased the ability of v-Abl to stimulate Ras activation and Erk phosphorylation in a Raf-dependent but Rac-independent fashion. Further analysis revealed that Shc is required for v-Abl-mediated Raf tyrosine 340 and 341 phosphorylation, an event associated with Erk phosphorylation. In contrast to effects on proliferation, survival of the cells and activation of Akt were not affected by expression of DN Shc. Together, these data reveal that v-Abl-Shc interactions are a critical part of the growth stimulatory signals delivered during transformation but that they do not affect antiapoptotic pathways. Furthermore, these data highlight a novel role for Shc in signaling from v-Abl to Raf.     

Mutations affecting the MA portion of the v-Abl protein reveal a conserved role of Gag in Abelson murine leukemia virus (MLV) and Moloney MLV

Abelson murine leukemia virus (Ab-MLV) arose from a recombination between gag sequences in Moloney MLV (Mo-MLV) and the c-abl proto-oncogene. The v-Abl oncoprotein encoded by Ab-MLV contains MA, p12, and a portion of CA sequences derived from the gag gene of Mo-MLV. Previous studies indicated that alteration of MA sequences affects the biology of Mo-MLV and Ab-MLV. To understand the role of these sequences in Ab-MLV transformation more fully, alanine substitution mutants that affect Mo-MLV replication were examined in the context of Ab-MLV. Mutations affecting Mo-MLV replication decreased transformation, while alanine mutations in residues dispensable for Mo-MLV replication did not. The altered v-Abl proteins displayed aberrant subcellular localization that correlated to transformation defects. Immunofluorescent analyses suggested that aberrant trafficking of the altered proteins and improper interaction with components of the cytoskeleton were involved in the phenotype. Similar defects in localization were observed when the Gag moiety containing these mutations was expressed in the absence of abl-derived sequences. These results indicate that MA sequences within the Gag moiety of the v-Abl protein contribute to proper localization by playing a dominant role in trafficking of the v-Abl molecule.     

v-src mutations outside the carboxyl-coding region are not sufficient to fully activate transformation by pp60c-src in NIH 3T3 cells.

Previous studies have shown that carboxyl-terminal mutation of pp60c-src can activate its transforming ability. Conflicting results have been reported for the transforming ability of pp60c-src mutants having only mutations outside its carboxyl-terminal region. To clarify the effects of such mutations, we tested the activities of chimeric v(amino)- and c(carboxyl)-src (v/c-src) proteins at different dosages in NIH 3T3 cells. The focus-forming activity of Rous sarcoma virus long terminal repeat (LTR)-src expression plasmids was significantly reduced when the v-src 3' coding region was replaced with the corresponding c-src region. This difference was masked when the Rous sarcoma virus LTR was replaced with the Moloney murine leukemia virus LTR, which induced approximately 20-fold more protein expression, but even focus-selected lines expressing v/c-src proteins were unable to form large colonies in soft agarose or tumors in NFS mice. This suggests that pp60c-src is not equally sensitive to mutations in its different domains and that there are at least two distinguishable levels of regulation, the dominant one being associated with its carboxyl terminus. v/c-src chimeric proteins expressed with either LTR had high in vitro specific kinase activity equal to that of pp60v-src but, in contrast, were phosphorylated at both Tyr-527 and Tyr-416. Total cell protein phosphotyrosine was enhanced in cells incompletely transformed by v/c-src proteins to the same extent as in v-src-transformed cells, suggesting that the carboxyl-terminal region may affect substrate specificity in a manner that is important for transformation.     

Active Akt and functional p53 modulate apoptosis in Abelson virus-transformed pre-B cells

Suppression of apoptosis is an important feature of the Abelson murine leukemia virus (Ab-MLV) transformation process. During multistep transformation, Ab-MLV-infected pre-B cells undergo p53-dependent apoptosis during the crisis phase of transformation. Even once cells are fully transformed, an active v-Abl protein tyrosine kinase is required to suppress apoptosis because cells transformed by temperature-sensitive (ts) kinase mutants undergo rapid apoptosis after a shift to the nonpermissive temperature. However, inactivation of the v-Abl protein by a temperature shift interrupts signals transmitted via multiple pathways, making it difficult to identify those that are critically important for the suppression of apoptosis. To begin to dissect these pathways, we tested the ability of an SH2 domain Ab-MLV mutant, P120/R273K, to rescue aspects of the ts phenotype of pre-B cells transformed by the conditional kinase domain mutant. The P120/R273K mutant suppressed apoptosis at the nonpermissive temperature, a phenotype correlated with its ability to activate Akt. Apoptosis also was suppressed at the nonpermissive temperature by constitutively active Akt and in p53-null pre-B cells transformed with the ts kinase domain mutant. These data indicate that an intact Src homology 2 (SH2) domain is not critical for apoptosis suppression and suggest that signals transmitted through Akt and p53 play an important role in the response.     

Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor.

Serum stimulates cells to increase their proportion of Ras protein in the active GTP-bound state. We have recently identified four types (I to IV) of apparently full-length cDNAs from a single mammalian gene, called CDC25Mm or GRF, which is homologous to the Ras-specific exchange factor CDC25 of S. cerevisiae. The largest cDNA (type IV) is brain specific, with the other three classes, although they have distinct 5' ends, essentially representing progressive N-terminal deletions of this cDNA. When placed in a retroviral expression vector, all four types of cDNAs induced morphologic transformation of NIH 3T3 cells and an increase in the basal level of GTP.Ras. Serum stimulation of these transformants lead to a further increase in GTP.Ras only in cells expressing the type IV cDNA. Each type of GRF protein was found in cytosolic and membrane fractions, and the protein in each fraction could stimulate guanine nucleotide release from GDP.Ras in vitro. When NIH 3T3 cells and cells expressing the type IV protein were transfected with two versions of a mutant ras gene, one encoding membrane-associated Ras protein and the other encoding a cytosolic Ras protein, the basal levels of GTP bound to both forms of the mutant Ras protein were significantly higher in the cells expressing the type IV protein. However, serum increased the level of GTP bound to the membrane-associated mutant Ras protein in NIH 3T3 cells and in cells expressing the type IV protein but not in cells expressing the cytosolic version of the Ras protein. We conclude that each type of CDC25Mm induces cell transformation via the ability of its C terminus to stimulate guanine nucleotide exchange on Ras, the presence of N-terminal sequences is associated with a serum-dependent change in GTP.Ras, and the serum-dependent increase in GTP.Ras by exogenous CDC25Mm or by endogenous exchange factors probably requires membrane association of both Ras and the exchange factor.     

Gag influences transformation by Abelson murine leukemia virus and suppresses nuclear localization of the v-Abl protein

Like the v-Onc proteins encoded by many transforming retroviruses, the v-Abl protein is expressed as a Gag-Onc fusion. Although the Gag-derived myristoylation signal targets the v-Abl protein to the plasma membrane, the protein contains the entire MA and p12 sequences and a small number of CA-derived residues. To understand the role of Gag sequences in transformation, mutants lacking portions of these sequences were examined for the effects of these deletions on v-Abl function and localization. Deletion of the N-terminal third of p12 or all of p12 enhanced the transformation of both pre-B cells and NIH 3T3 cells. In contrast, deletions in MA or a deletion removing all of Gag except the first 34 amino acids important for myristoylation highly compromised the ability to transform either cell type. Although all of the mutant proteins retained kinase activity, those defective in transformation were reduced in their ability to activate Erk, suggesting a role for Gag sequences in v-Abl signaling. Immunofluorescence analysis revealed that a v-Abl protein retaining only the first 34 amino acids of Gag localized to the nucleus. These data indicate that Gag sequences are important for normal v-Abl signaling and that they suppress nuclear localization of the molecule.     

p53 Mediates Apoptotic Crisis in Primary Abelson Virus-Transformed Pre-B Cells

Transformation of pre-B cells by Abelson murine leukemia virus (Ab-MLV) involves a balance between positive, growth-stimulatory signals from the v-Abl oncoprotein and negative regulatory cues from cellular genes. This phenomenon is reflected by the clonal selection that occurs during Ab-MLV-mediated transformation in vivo and in vitro. About 50% of all Ab-MLV-transformed pre-B cells express mutant forms of p53 as they emerge from this process, suggesting that this protein may play an important role in the transformation process. Consistent with this idea, expression of p19(Arf), a protein whose function depends on the presence of a functional p53, is required for the apoptotic crisis that characterizes primary Ab-MLV transformants. To test the role of p53 in pre-B-cell transformation directly, we examined the response of Trp53(-/-) mice to Ab-MLV. The absence of p53 shortens the latency of Abelson disease induction but does not affect the frequency of cells susceptible to Ab-MLV-induced transformation. However, primary transformants derived from the null animals bypass the apoptotic crisis that characterizes the transition from primary transformant to fully malignant cell line. These effects do not require p21(Cip-1), a major downstream target of p53; however, consistent with a role of p19(Arf), transformants expressing mutant p53 and abundant p19 retain wild-type p19 sequences.     

Temperature-sensitive mutants of Abelson murine leukemia virus deficient in protein tyrosine kinase activity.

The effect of two missense mutations in abl on transformation by Abelson murine leukemia virus was evaluated. These mutations led to the substitution of a histidine for Tyr-590 and a glycine for Lys-536. Both changes gave rise to strains that were temperature dependent for transformation of both NIH 3T3 cells and lymphoid cells when expressed in the context of a truncated Abelson protein. In the context of the prototype P120 v-abl protein, the Gly-536 substitution generated a host range mutant that induced conditional transformation in lymphoid cells but had only a subtle effect on NIH 3T3 cells. The combination of both substitutions gave rise to a P120 strain that was temperature sensitive for both NIH 3T3 and lymphoid cell transformation. The Abelson proteins encoded by the temperature-sensitive strain displayed in vitro kinase activities that were reduced when compared with those of wild-type proteins. In vivo, levels of phosphotyrosine were reduced only at the restrictive temperature. Analysis of cells expressing either the wild-type P160 v-abl protein or the P210 bcr/abl protein and an Abelson protein encoded by a temperature-sensitive strain failed to correct this defect, suggesting either that tyrosine phosphorylation in vivo is an intramolecular reaction or that the protein encoded by the temperature-sensitive strain is a poor substrate for tyrosine phosphorylation in vivo. These results raise the possibility that tyrosine phosphorylation of Abelson protein plays a role in transformation.     

The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein.

Although the Rap1A protein resembles the oncogenic Ras proteins both structurally and biochemically, Rap1A exhibits no oncogenic properties. Rather, overexpression of Rap1A can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and Rap1A occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of Rap1A were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of Rap1A) in NIH 3T3 cells and a full-length human Rap1A protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length Rap1A, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid. In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the Rap1A COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:Rap1A protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of Rap1A can support the interactions between H-Ras and membranes that are required for transformation.     

The transforming potential of the c-erbB-2 protein is regulated by its autophosphorylation at the carboxyl-terminal domain.

The mutant c-erbB-2 protein with Glu instead of Val-659 exhibited transforming activity in NIH 3T3 cells. This protein showed enhanced tyrosine kinase activity in vitro and enhanced autophosphorylation at Tyr-1248 located proximal to the carboxyl terminus. Enhanced tyrosine phosphorylation of several cellular proteins was detected in cells expressing the Glu-659 c-erbB-2 protein. Introduction of an additional mutation at the ATP-binding site (Lys-753 to Met) of this protein resulted in abolition of its transforming ability. These data indicate that the transforming potential of c-erbB-2 is closely correlated with elevated tyrosine kinase activity of the gene product. To investigate the role of autophosphorylation in cell transformation, we introduced an additional mutation at the autophosphorylation site of the Glu-659 c-erbB-2 protein (Tyr-1248 to Phe). This mutant protein exhibited lower tyrosine kinase activity and lower transforming activity. On the other hand, when the carboxyl-terminal 230 amino acid residues were deleted from the c-erbB-2 protein, the tyrosine kinase activity and cell-transforming activity of the protein were enhanced. Thus, the carboxyl-terminal domain, which contains the major autophosphorylation site, Tyr-1248, may regulate cellular transformation negatively and autophosphorylation may eliminate this negative regulation.     

Distal end of carboxyl terminus is not essential for the assembly of rat Eag1 potassium channels

The assembly of four pore-forming α-subunits into tetramers is a prerequisite for the formation of functional K(+) channels. A short carboxyl assembly domain (CAD) in the distal end of the cytoplasmic carboxyl terminus has been implicated in the assembly of Eag α-subunits, a subfamily of the ether-à-go-go K(+) channel family. The precise role of CAD in the formation of Eag tetrameric channels, however, remains unclear. Moreover, it has not been determined whether other protein regions also contribute to the assembly of Eag subunits. We addressed these questions by studying the biophysical properties of a series of different rat Eag1 (rEag1) truncation mutants. Two truncation mutants without CAD (K848X and W823X) yielded functional phenotypes similar to those for wild-type (WT) rEag1 channels. Furthermore, nonfunctional rEag1 truncation mutants lacking the distal region of the carboxyl terminus displayed substantial dominant-negative effects on the functional expression of WT as well as K848X and W823X channels. Our co-immunoprecipitation studies further revealed that truncation mutants containing no CAD indeed displayed significant association with rEag1-WT subunits. Finally, surface biotinylation and protein glycosylation analyses demonstrated that progressive truncations of the carboxyl terminus resulted in aggravating disruptions of membrane trafficking and glycosylation of rEag1 proteins. Overall, our data suggest that the distal carboxyl terminus, including CAD, is dispensable for the assembly of rEag1 K(+) channels but may instead be essential for ensuring proper protein biosynthesis. We propose that the S6 segment and the proximal carboxyl terminus may constitute the principal subunit recognition site for the assembly of rEag1 channels.     

The amino and carboxyl termini of perilipin a facilitate the storage of triacylglycerols

Perilipin A is the most abundant lipid droplet-associated protein in adipocytes and serves important functions in regulating triacylglycerol levels by reducing rates of basal lipolysis and facilitating hormonally stimulated lipolysis. We have previously shown that the central region of perilipin A targets and anchors it to lipid droplets, at least in part via three moderately hydrophobic sequences that embed the protein into the hydrophobic core of the droplet. The current study examines the roles of the amino and carboxyl termini of perilipin A in facilitating triacylglycerol storage. Amino- and carboxyl-terminal truncation mutations of mouse perilipin A were stably expressed in 3T3-L1 preadipocytes, which lack perilipins. Triacylglycerol content of the cells was quantified as a measure of perilipin function and was compared with that of cells expressing full-length perilipin A or control cells lacking perilipins. The amino-terminal sequence between amino acids 122 and 222, including four 10-11-amino acid sequences predicted to form amphipathic beta-strands and a consensus site for cAMP-dependent protein kinase, and the carboxyl terminus of 112 amino acids that is unique to perilipin A were critical to facilitate triacylglycerol storage. The precocious expression of full-length perilipin A in 3T3-L1 preadipocytes aided more rapid storage of triacylglycerol during adipose differentiation. By contrast, the expression of highly truncated amino- or carboxyl-terminal mutations of perilipin failed to serve a dominant negative function in lowering triacylglycerol storage during adipose differentiation. We conclude that the amino and carboxyl termini are critical to the function of perilipin A in facilitating triacylglycerol storage.     

Regulation of Sos Activity by Intramolecular Interactions

The guanine nucleotide exchange factor Sos mediates the coupling of receptor tyrosine kinases to Ras activation. To investigate the mechanisms that control Sos activity, we have analyzed the contribution of various domains to its catalytic activity. Using human Sos1 (hSos1) truncation mutants, we show that Sos proteins lacking either the amino or the carboxyl terminus domain, or both, display a guanine nucleotide exchange activity that is significantly higher compared with that of the full-length protein. These results demonstrate that both the amino and the carboxyl terminus domains of Sos are involved in the negative regulation of its catalytic activity. Furthermore, in vitro Ras binding experiments suggest that the amino and carboxyl terminus domains exert negative allosteric control on the interaction of the Sos catalytic domain with Ras. The guanine nucleotide exchange activity of hSos1 was not augmented by growth factor stimulation, indicating that Sos activity is constitutively maintained in a downregulated state. Deletion of both the amino and the carboxyl terminus domains was sufficient to activate the transforming potential of Sos. These findings suggest a novel negative regulatory role for the amino terminus domain of Sos and indicate a cooperation between the amino and the carboxyl terminus domains in the regulation of Sos activity.