Compartmentalized signal transduction by receptor tyrosine kinases

Communication between cells and the ability to respond to environmental changes is essential for the regulation of cell metabolism, proliferation and differentiation. Many of these signals are transmitted by receptor tyrosine kinases (RTKs). On binding ligand, the autophosphorylation state of the kinase is altered and this change is detected within the cell as an altered association of the cytoplasmic portion of the receptor with intracellular signalling molecules. These form part of signalling cascades that feed the information from the receptor into the nucleus, where changes in gene expression are induced. Many of these pathways have been described in great detail, but one aspect of these signalling pathways has largely been overlooked. It has been known for some time that RTKs become rapidly internalized on binding their ligands and, generally, this is assumed to be a way of downregulating cellular responses. However, there is accumulating evidence that the internalized receptors continue to signal within the endosome and that this could be an important facet of their behaviour.

Compartmentalized signal transduction by receptor tyrosine kinases

Signal transduction through receptor tyrosine kinases is believed to occur mainly at the plasma membrane. Ligands bind to their cognate receptors and trigger autophosphorylation

events, which

are detected by intracellular signalling molecules. However, ligands, such as epidermal growth factor and insulin, induce the

Signalling

at the plasma

membrane

RTKs consist of an extracellular ligand-binding domain, a transmembrane region and a cytoplasmic portion containing a catalytic kinase domain. Ligand binding induces receptor dimerization, kinase activation and the transphosphorylation of the receptor on defined tyrosine residues. Progress in understanding the mechanisms of RTK signal transduction has been made with the identification of multiple downstream targets of activated receptors’. Many of these proteins contain Src homology region 2 (SH2) domains and/or phosphotyrosine-binding (PTB) domains that specifically recognize phosphorylated tyrosine residues within the context of the flanking amino acids2. These specific interactions define the signal transduction pathways accessible to each activated, phosphorylated receptor. The tyrosine autophosphorylation of the epidermal growth factor receptor (EGF-R) at specific sites leads to the recruitment and stable association of several downstream signalling molecules (Fig. la). One SH2-containing protein, growth factor receptor binding protein 2 (GRB2), binds to phosphotyrosine 1068 of the EGF-R (Ref. 1). GRB2 acts as an adaptor protein and brings mSOS (the mammalian homologue of SON OF SEVENLESS), the GRBZ-associated GDP-GTP exchange protein, to ~21”’ (Ref. 3). This results in the GTP activation of ~21”~ and the subsequent activation of the cytosolit mitogen-activated protein (MAP) kinase cascade4. The EGF-R-associated adaptor protein SHC has been proposed to act in an analogous manner. SHC contains both a PTB and a SH2 domain, enabling it to bind two phosphorylated tyrosine residues of the EGF-R (Tyr1148 and Tyr1173, respectively)5. Unlike GRB2, SHC is also a substrate for the activated EGF RTK. Phosphorylation of SHC on Tyr317 provides a site for the binding of the GRB2 SH2 domain and thus serves as a further mechanism for recruitment of mSOS to plasma-membrane-associated ~21” (Fig. la; Refs 1 and 2). TRENDS

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rapid internalization

of their receptors into endosomes. Although

this event is traditionally

thought to attenuate the ligand-induced

response, in this article the authors discuss an alternative scenario in which selective and regulated signal transduction from receptor tyrosine kinases occurs within the endosome.

A different scenario for signal transduction from the insulin receptor (I-R) has been proposed. Although insulin induces both activation of the kinase function of its receptor and the transphosphorylation of the I-R cytosolic tail, the stable association of adaptor proteins for the receptor has been more difficult to establish. Nevertheless, the major substrate of the I-R kinase, insulin receptor substrate1 (IRS-l), is phosphorylated in response to insulin stimulation. The multiple tyrosine phosphorylated residues of IRS-l act as docking sites for a number of signal-transducing molecules, including the lipid kinase, phosphatidylinositol 3-kinase (PI 3-kinase)6. PI 3-kinase is activated as a consequence of the binding of the two SH2 domains of its ~85 regulatory subunit to phosphotyrosine residues of IRS-l, all of which are found within YXXM motifs6r7. The major in vivo substrate for the catalytic 1 lo-kDa subunit of PI 3-kinase is probably membrane-bound phosphatidylinositol (4,5)-bisphosphate, from which is generated phosphatidylinositol(3,4,5)-trisphosphate (Ref. 8). The use of inhibitors of PI 3-kinase has indicated that several metabolic actions attributed to the activated insulin RTK involve activation of this lipid kinase9jr0. Ligand-mediated

receptor

internalization

As indicated in Figure l(a), signal transduction pathways appear to be initiated at the plasma 0 1995 Elsevier Science Ltd

Patricia Baass and John Bergeron (Dept of Anatomy and Cell Biology), G. M. Di Cuglielmo (Dept of Biochemistry) and Barry Posner (Dept of Medicine) are at McGill University, Montreal, P. Q., Canada H3A 282; and Frarqois Authier is at the INSERM U30, HBpital des Enfants Malades, Paris, France.

46.5

EGF-R

Plasma membrane

GRB2

SHC

Signal transduction

3

Signal transduction

\

(b)

Internalization \ Signal transduction

(c) Time (min) after injection

Plasma membrane 0

0.5

5

15

Endosomes 30

60

0

0.5

5

15

30

60

-+- EGF-R (170 kDa) +-- I-R (94 kDa)

Insulin

membrane. However, in the major in vivo organ enriched in receptors for EGF and insulin (liver parenchyma), the binding of the cognate ligands induces the rapid internalization of their respective receptors into endosomes without an appreciable time-lag (Figs lb and lc; Ref. 11). For the EGF receptor, internalization may represent a desensitization and/or attenuation response since the rapid sequestration of the receptor into endosomes removes it from its downstream target, p21ras, the majority of which is constitutively associated with the plasma membrane12. Indeed, the introduction of truncated EGF RTKs, which retained kinase activity but were internalization-defective, into NR6 cells led to enhanced mitogenesis and cellular transformation 466

implying a relationship between internalization and attenuationi3. In the case of the I-R, the rapid internalization may not be linked to attenuation since its major substrate, IRS-l, is a soluble protein6. However, in endosomes of liver parenchyma, EGFand insulin-RTK signalling appears to be regulated selectively (Fig. lb); this may be the case for other receptor tyrosine kinases too. Endosomal regulation the EGF receptor

of signal

transduction

-

We found that internalized EGF-R kinase is highly tyrosine phosphorylated in endosomes of rat liver”. This state has also been visualized directly by immunoelectron microscopy of A431 cells overexpressing TRENDS

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FIGURE

1

Ligand-mediated receptor activation. (a) internalization and compartmentalization. Ligand binding induces a conformational change in the receptor and consequent receptor transactivation. In the case of the epidermal growth factor receptor (EGF-R), ligand-mediated receptor dimerization and kinase activation lead to the autophosphorylation of Tyr1068, which serves as a site of recruitment for the Src homology region 2 (SH2) domain of the adaptor protein GRB2. The two SH3 domains of CRB2 interact with the proline-rich sequences of the guanine-nucleotide exchange factor mSOS. The direct physical interaction of mSOS with ~21 rus may promote ~21”’ activation and signal transduction via a MAP kinase phosphorylation cascade. The EGF-R is also phosphorylated on other Tyr residues that can recruit the adaptor protein SHC. When SHC is itself tyrosine phosphorylated by the ECF-R kinase, it acts as a further link between GRB2-mSOS and p21 w activation. Insulin receptor (I-R) activation is a consequence of ligand-mediated receptor autophosphorylation. A major substrate of the I-R, insulin receptor substrate-l (IRS-l), is Tyr-phosphorylated after transient binding to the receptor, resulting in a stable association with the SH2 domains of the ~85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase). This leads to the activation of the associated catalytic subunit of PI 3-kinase and subsequent wortmannin-sensitive insulin-mediated signalling. Asterisks indicate the Tyr-phosphorylated residues. (b) Ligand-mediated receptor internalization. Upon interaction with their respective ligands, the ECF-R and I-R both internalize into endosomes without a noticeable time-lag and with similar initial half-times of -1 min. For the ECF-R, maximal Tyr-phosphorylation of both the receptor and SHC and the increased recruitment of GRB2 and mSOS to the activated receptor are observed in endosomes. There is also a cytosolic pool of activated, phosphorylated SHC-CRB2-mSOS, probably originating from the endosome, which may extend p21 r0s activation beyond that restricted to the plasma membrane EGF-R. For the I-R, its degree of phosphorylation decreases in the endosome, whereas its kinase activity increases compared to that at the plasma membrane. The endosomal insulin receptor tyrosine kinase (RTK) can phosphorylate IRS-l, but the altered pattern of phosphorylation of the former may affect this function and redefine the array of molecules the I-R can interact with after internalization. (c) The rapid internalization of receptors is revealed in immunoblots of the ECF-R and the 94-kDa B-subunit of the I-R evaluated in plasma membrane (100 pg protein) and endosomal fractions (50 pg protein) isolated from rat liver homogenates at the indicated times after systemic injection with receptor-saturating doses of ligand, as described in Ref. 11.

EGF-Rs14.The cytosolic orientation of the tyrosinephosphorylated tail and the presence of an active EGF RTK in endosomes over a prolonged period of time suggests that the receptor may continue to signal after internalization (Fig. lb; Refs 15 and 16). Although SHC bound initially to activated EGF RTK at the plasma membrane, tyrosine phosphorylated SHC was found mainly in the endosome. In fact, a complex of activated EGF RTK, tyrosine phosphorylated SHC and GRBZ-mSOS was found for an extended period of time in the endosomal TRENDS

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membrane. This coincided with the appearance of a cytosolic pool of tyrosine-phosphorylated SHC associated with GRB2-mSOS (Ref. 11). Since ~21” was activated over a similar time-course, then, first, a rationale for the adaptor protein SHC was proposed in which it serves as a cytosolic intermediate in signal transduction and, second, the endosome was the likely source of this pool of activated cytosolic SHC at later times, that is, after EGF-R internalization. Such a scenario would serve as an effective amplification mechanism for accessing p21ras, over and above the subset that is in physical association, via adaptor proteins, with plasma-membrane-bound EGF-R. The endosomal apparatus is positioned both temporally and physically in the pathway between the plasma membrane and the lysosome. The endosomal lumen becomes progressively more acidic and provides an environment expected to cause the dissociation of internalized ligand-receptor complexesi7. Although EGF is resistant to dissociation from its receptor in endosomes15, this is not the case for other EGF-R ligands l* . Transforming growth factor 01 (TGFa), a highly potent EGF agonist, has an identical affinity constant for binding to the EGF-R, but dissociates at a markedly higher pH (half-maximal dissociation at pH 6.9) as evaluated in murine B82L cells expressing the human EGF-R or in human keratinocytes with endogenous EGF RTK. Although both EGF and TGFcx have similar internalization kinetics, the targeting of the receptor for degradation in lysosomes (downregulation) was achieved more effectively by EGF than by TGFo((Ref. 19). The greater biological potency of TGFa may be due to the repeated presentation of recycled receptors for TGFcY and EGF at the cell surface, resulting in several rounds of signalling and, in this way, the occupancy-induced downregulation of the EGF-R by EGF is bypassedzO. Whether the tyrosine phosphorylation of the EGF-R is enhanced in the endosome following TGFamediated receptor internalization is unknown. Selective ligand dissociation may be an additional, endosomally located, mechanism further regulating signal transduction from the EGF-R. The PDGF receptor

The ability of endosomal platelet-derived growth factor receptor (PDGF-R) to perform signal transduction is supported by several observations. In 3T3-Ll cells, the prolonged time-course of tyrosine phosphorylation of the receptor suggests that it enters the endocytic pathway in a catalytically active form2i. In porcine endothelial cells expressing the PDGF-R, the pH-dependence of homodimeric PDGF-BB dissociation from the receptor revealed that, even in the acidic environment of the endosome, little ligand dissociation occurredz2, enabling the receptor to retain its kinase activity and signalling capability after internalization. Signal transduction from the activated PDGF-R is carried out by PI 3-kinase in addition to several of the adaptor molecules used by the EGF RTK. (Ref. 23). The presence of PI 3-kinase activity in clathrin-coated vesicles (which represent the initial internalization event) of PDGF-stimulated

467

cells suggests that active PI 3-kinase is internalized along with the receptor. In contrast to the more transient effects of PDGF, the formation of D-3phosphorylated lipids is sustained for 15-30 min, a time-course that parallels the time required for the activated receptors (presumably associated with active PI 3-kinase) to move from the plasma membrane to, and through, the endosomal apparatus21~22. The NCF receptor

(trk A)

Nerve growth factor (NGF) signal transduction involves the activation of a RTK, trk A, and the tyrosine phosphorylation of SHC, the activation of phospholipase C-y1 and PI 3-kinase, and activation of both ~21”~ and a MAP kinase cascadei. In sciatic nerve axons in vivo, NGF-mediated internalization of trk A into endosomes coincided with enhanced tyrosine phosphorylation of trk A, which increased as receptor internalization progressed into the more distal segments of the axon24. Although internalization has been linked to trk A kinase activation, downstream signalling from the internalized activated trk A awaits elucidation. Endosomal

p60 c-Src and endosomal

Rho B

Src is the prototype tyrosine kinase, which was originally linked to growth control as deduced from the transforming activity of mutant v-Src (Ref. 25). A role for c-Src in integrin-mediated signal transduction has recently been elucidated in NIH 3T3 cells2’j. An association of activated c-Src with focal adhesion tyrosine kinase (FAK) at cytosolic sites coinciding with cell attachment to the extracellular surface is proposed to cause the tyrosine phosphorylation of FAK and activation of the Ras signal-transduction pathway via the recruitment of adaptor proteins. However, in fibroblasts, c-Src is normally located in the endosomal membrane27. Induction of the integrin-mediated signalling pathway coincides with the dephosphorylation of the negative-regulatory Tyr527 of endosomal c-Src. This coincides with increased kinase and autophosphorylation activity of c-Src in the endosome prior to migration of the kinase to focal adhesions at the plasma membranez8. Like c-Src, the Ras-related GTP-binding protein Rho B is constitutively localized to endosomes after expression in rat-2 fibroblasts, Cos-1 and MDCK cells2’. Although Rho B is biologically active in these cells (it stimulates actin stress fibre assembly at focal adhesions), the relevance of the endosomal location is unknown. The insulin

receptor

In rat liver parenchymal cells, I-R internalization is accompanied by a decrease in phosphotyrosine content as compared to the ligand-induced phosphotyrosine state at the plasma membrane11,30. However, detailed biochemical studies revealed an increase in the insulin RTK activity in endosomes (i.e. the activated V,,,/K, of tyrosine phosphorylation of in vitro substrates; Ref. 30). These observations are analogous to those made in the case of c-Src (above) where the selective dephosphorylation of its 468

negative-regulatory tyrosine resulted in kinase activation. As for c-Src, the presence of endosomal tyrosine phosphatases for the I-R was inferred. Indeed, protein phosphotyrosine phosphatase activities have been demonstrated on the cytosolic side of endosome membranes31. Initial studies failed to define specificity in the ability of the phosphatase to distinguish between internalized receptors for EGF and insulin, although specificity was inferred since the phosphotyrosine content of internalized EGF RTK increases, while that of the I-R decreases16T30. We have found recently that the introduction in vivo of the potent phosphotyrosine phosphatase inhibitor bisperoxo (l,lO-phenanthroline) oxovanadate anion [bpV(phen); Ref. 32; Fig. 21 had a selective effect on the phosphotyrosine content of the basal level of I-R constitutively present in endosomes33. Augmentation of the phosphotyrosine content of the endosomal insulin RTK by bpV(phen) led to the enhanced phosphorylation of IRS-l as well as to other downstream effects. In fat cells, activated tyrosine-phosphorylated I-Rs have been observed in endosomes after ligand-mediated internalization and have been implicated in the tyrosine phosphorylation of IRS-l induced by insulin in these cells34. An intracellular compartment containing tyrosine-phosphorylated IRS-l complexed to activated PI 3-kinase has been observed in fat cells after insulin administration, although this compartment was not physically coincident with endosomes containing internalized I-Rs~~. However, the coincident time-courses of activation of the insulin RTK in the endosome and the appearance of tyrosine phosphorylation of IRS-l in the intracellular compartment are suggestive of a causal link34. A major physiological consequence of insulin activation of fat cells (and muscle) is the increase in V,, of glucose transport activity at the plasma membrane. In the basal state, glucose transporters are normally located in incompletely defined intracellular compartments 36~37.After insulin administration, glucose transporters (GLUT-4) from these compartments egress by means of vesicle transport to the fatcell plasma membrane, thereby increasing the number of cell surface transporters and hence the V,, of glucose transport. More specifically, insulin induces GLUT-4 recycling through the same endosomal pathway that internalizes activated insulin RTK. However, no colocalization between recycling I-Rs and GLUT- 4 vesicles has been demonstrated and, furthermore, no causal link between the internalized insulin RTK signal transduction and GLUT-4 has been shown. Another possible mechanism of regulation of I-R signal transduction in the endosome may reside in the endosomal lumen of liver parenchyma. Here, a potent proteinase was identified that binds and degrades insulin in acidic conditions, and this could act to terminate ligand-mediated restimulation of insulin RTK activity 38.This proteinase is unrelated to other endosomal proteinases identified and characterized thus far39. The distribution of proteinases in early and late endosomes, as well as the decreasing pH gradient, may be indicative of selective mechanisms for degrading incoming ligands, with insulin TRENDS

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being the most susceptible ligand to date (Refs 38,39; Fig. 2).

Cytoplasm pH 7.2

/

““‘“A

The IGF-1 receptor

Rat-l fibroblasts in culture express high levels of insulin-like growth factor-l (IGF-1) receptors, which are similar in structure to I-Rs, but whose functions are growth related rather than metabolic40. In a recent study, the kinetics of IGF-1 internalization in rat-l fibroblasts were compared with those of insulin in the same cells overexpressing the I-R. A marked difference in the endosomal dissociation of the cognate ligands was uncovered. Whereas insulin was dissociated and degraded rapidly after internalization, IGF-1 was more resistant to acid-induced dissociation from its receptor, resulting in a prolonged (up to 120 min) accumulation of intracellular (endosomal) IGF-1 (Ref. 40). The differences in endosomal ligand dissociation have been speculated to regulate the different bioeffects of the two related receptors. Signai

transduction

and sorting

Ligand-mediated internalization of RTKsis dependent on the receptor-mediated tyrosine phosphorylation of a substrate(s)41, which may be the RTK cytosolic tail. Aromatic amino acids in the context of a tight-turn motif have been found in the cytosolic tails of constitutively internalizing nutrient receptors and RTKs. This motif has been causally linked to the ability to access the endocytic machinerp2. For the nutrient receptors [low-density lipoprotein (LDL) receptor and LDL receptor-related protein (LRP)], this motif corresponds to NPXY. These motifs in cytosolic tails of RTKs are causally linked to signal transduction2, although site-specific mutagenesis has failed to establish a definite link between tyrosine phosphorylation of RTK NPXY and accessto the endocytic apparatus 43,44.Tyrosine-phosphorylation-dependent association of the clathrin-associated protein complex AP2 with the EGF RTK is an attractive candidate for effecting EGF-R sorting to the endocytic apparatus45. Furthermore, dynamin, another constituent of the endocytic apparatus46, has been shown to interact with GRB2 and phospholipase C-y and has also been implicated in a mechanism for EGF RTK accessto the endocytic apparatus47. In the case of the I-R, a novel protein, enigma, has been found by the yeast two-hybrid system to associate with insulin RTK polypeptide sequences containing the motifs (GPLY953 and NPEY960) implicated in accessing the endocytic apparatus 48. For one of these motifs, phosphotyrosine 960 is the site of recruitment of IRS-l (Ref. 49). However, further work is required for the establishment of a causal link that connects these proteins and RTK internalization. The proper sorting of the PDGF-R into endosomes has been shown to be dependent on the tyrosine phosphorylation of the cytoplasmic tail at sites (Tyr.540 and Tyr.541) that bind PI 3-kinase50. However, other RTKs, the I-R for example, do not contain such domains. Clearly, a missing molecular link(s) between signal transduction and RTK internalization awaits elucidation and may be clarified in the general context of endocytic code-dependent association TRENDS

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I-R

EGF-R

#

ATi’ FIGURE

IH+

u

PTPase

ADP

2

Endosomal regulation of selective ligand degradation and receptor tyrosine kinase (RTK) dephosphorylation. There is little dissociation or degradation of epidermal growth factor (E) within hepatic endosomes. However, an acid-pH-coupled dissociation and degradation of insulin (I) is effected via endosomal acidic insulinase (EAI). The pH of endosomes is regulated by an electrogenic proton-pumping ATPase. RTK phosphotyrosine phosphatases (PTPase) are highly active in endosomes, and the endosomal insulin RTK is selectively targeted and activated owing to inhibition of its PTPase by the potent peroxovanadium PTPase inhibitor [bpV(phen)]. Asterisks indicate the Tyr-phosphorylated residues. EGF-R, epidermal growth factor receptor; I-R, insulin receptor.

of unknown adaptor proteins that bind both to nutrient receptors and to RTKs. While receptors such as those for EGF and PDGF are targeted largely to lysosomes for degradation, others such as the RTKs for insulin and IGF-1 are recycled largely to the plasma membrane17. Hence, a sorting machinery must be in the endosome to sort different receptors into different vesicle populations. Endosomal RTK tyrosine phosphorylation of annexin 1, a modulator of membrane structure, has been proposed as the mechanism by which the EGF-R at the periphery of the endosomal membrane is sorted into the inner vesicles of multivesicular endosomes and then subsequently targeted for degradation in lysosomes 51. The further identification of RTK phosphorylated molecules in endosomes may not only uncover the missing molecular links in receptor traffic, but also extend our knowledge of further mechanisms for the propagation of signal transduction. Indeed, the selection of differentiation or metabolic pathways, asopposed to pathways involved in cell cycle and growth control, for different RTKs may be a consequence of their compartmentalization. The availability of a growing number of reagents for studying RTK signal transduction, membrane traffic and subcellular compartments suggests that this is likely to be an expanding field of study. References 1

VAN DERCEER,

P., HUNTER, T. and LINDBERG, R. A. (1994) Annu. Rev. Cell Do/. 10, 251-337 2 VAN DERCEER, P. and PAWSON, T. (1995) Trends Biochem. Sci. 20, 277-280 3 BUDAY, L. and DOWNWARD, J. (1993) Cell 73,61 I-620

469

Acknowledgements P. Baass is supported by a studentship from the FCAR of Quebec. C. M. Di Cuglielmo is supported by a Steve Fonyo studentship of the NCI of Canada. The work is supported by operating grants from the NCI and MRC of Canada to B. I. Posner and 1.1. M. Bergeron. Owing to space limitations, several ideas discussed are represented inadequately in the references. This paper is dedicated to the memory of James Jay Doherty II.

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4 SECER, R. and KREBS, E. C. (1995) FASEB 1.9, 726-735 5 BATZER, A. G., BLAIKIE, I’., NELSON, K., SCHLESSINGER, J. and MARGOLIS, B. (1995) Mol. Cell. Viol. 15,4403-4409 6 MYERS, M. G., SUN, X. 1. and WHITE, M. F. (1994) Trends Biocbem. Sci. 19, 289-293 7 BACKER, J. et al. (1992) EMBO]. 11, 3469-3479 8 STEPHENS, L. (1995) Biochem. Sot. Trans. 23,207-221 9 CHEATHAM, B., VLAHOS, C. I., CHEATHAM, L., WANG, L., BLENIS, 1. and KAHN, C. R. (1994) Mol. Cell. Biol. 14, 4902-4911 10 OKADA, T., KAWANO, Y., SAKAKIBARA, T., HAZEKI, 0. and UI, M. (1994) 1. Biol. C/rem. 269,3568-3573 11 DI GUGLIELMO, G. M., BAASS, P. C., OU, W-j., POSNER, B. I. and BERGERON, 1.1. M. (1994) EMBO /. 13,4269-4277 12 BAR-SAGI, D., SUHAN, I., MCCORMICK, F. and FERAMISCO, J. R. (1988) 1. Cell Biol. 106, 1649-l 658 13 WELLS, A., WELSH, 1. B., IAZAR, C. S., WILEY, H. S., GILL, G. N. and ROSENFELD, M. G. (1990) Science 247,962-964 14 CARPENTIER, J-L., WHITE, M. F., ORCI, L. and KAHN, R. C. (1987) /. CellBiol. 105, 2751-2762 15 LAI, W. H., CAMERON, P. H., DOHERTY II, I-J., POSNER, B. I. and BERGERON, 1. J. M. (1989) 1. Cell Biol. 109, 2751-2760 16 WADA, I., LAI, W. H., POSNER, B. I. and BERGERON, 1. J. M. (1992) 1. Cell Biol. 116, 321-330 17 BERGERON, 1. J. M., CRUZ, J., KHAN, M. N. and POSNER, B. I. (1985) Annu. Rev. Physiol. 47, 383403 18 FRENCH, A. R., TADAKI, D. K., NIYOGI, 5. K. and LAUFFENBURGER, D. A. (1995) 1. Biol. C/rem. 270,4334-4340 19 EBNER, R. and DERYNCK, R. (1991) Cell Regul. 2,599-612 20 FRENCH, A. R., SUDLOW, G. P., WILEY, H. 5. and LAUFFENBURGER, D. A. (1994) 1. Biol. Chem. 269, 15749-1575s 21 KAPELLER, R., CHAKRABARTI, R., CANTLEY, L., FAY, F. and CORVERA, S. (1993) Mol. Cell. Biol. 13, 6052-6063 22 SORKIN, A., ERIKSSON, A., HELDIN, C-H., WESTERMARK, B. and CLAESSON-WELSH, L. (1993) 1. Cell. Physiol. 156, 373-382 23 CLAESSON-WELSH, L. (1994) /. Biol. Chem. 269, 32023-32026 24 EHLERS, M. D., KAPLAN, D. R., PRICE, D. L. and KOLIATSOS, V. E. (1995) 1. Cell Biol. 130, 149-156 25 COOPER, 1. A. (1989) in Peptides and Protein Phosphorylotion (Kemp, B. ed.), pp. 85-l 13, CRC Press 26 SCHAEPFER, D. D., HANKS, 5. K., HUNTER, T. and VAN DER GEER, P. (1994) Nature 372, 786-791 27 KAPLAN, K. B., SWEDLOW, 1. R., VARMUS, H. E. and MORGAN, D. 0. (1992) /, Cell Biol. 118, 321-333

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