MINIREVIEW Coordinated action of protein tyrosine phosphatases in insulin signal transduction Alan Cheng, Nadia Dube ´ , Feng Gu and Michel L. Tremblay Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Quebec, Canada Insulin is the principal regulatory hormone involved in the tight regulation of fuel metabolism. In response to blood glucose l evels, it is secrete d by the b cells o f t he pancreas and exerts its effects by binding to cell surface receptors that are present on virtually all cell types and tissues. In humans, perturbations in insulin function and/or secretion lead to diabetes mellitus, a severe disorder primarily characterized by an inability to maintain blood glucose homeostasis. Furthermore, it is estimated that 90–95% of diabetic patients exhibit resistance to insulin action. Thus an understanding of insulin signal transduction and insulin resistance at the molecular level is crucial to the understanding of the patho- genesis of this disease. The in sulin receptor (IR) is a trans- membrane tyrosine kinase that becomes activated upon ligand binding. Consequently, the receptor and its down- stream substrates become tyrosine p hosphorylated. This activates a series of intracellular signaling cascades which coordinately initiate the appropriate biological response. One important mechanism by which insulin signalin g is regulated involves the protein t yrosine phosphatases (PTPs), which may either act on the IR itself and/or its substrates. Two w ell characterized examples include leuckocyte antigen related (LAR) and protein tyrosine phosphatase-1B (PTP- 1B). The present review will discuss the current knowledge of these two and othe r potential PTPs involved in the insulin signaling pathway. Keywords: diabetes; insulin receptor; protein tyrosine phos- phatase; knockout mice; signaling. INTRODUCTION Insulin is the most potent anabolic hormone identified to date. It is produced and secreted in a regulated fashion by the b cells in the pancreatic islet. Practic ally all cell types are responsive to insulin, although the term Ôinsulin sensitive tissuesÕ often r efer to the liver, muscle a nd adipose. The primary biological effect of insulin is to maintain glucose homeostasis. It acutely promotes glucose uptake in muscle and adipose tissue, while suppressing hepatic glucose production. However, in sulin also stimulates lipogenesis, protein synthesis, and has been shown to be a mitogen for certain cell types. The importance of insulin function is highlighted by its disregulation in diabetes mellitus, a human disease charac- terized by an impairment in insulin secretion (type I; insulin dependent) and/or action (type II; noninsulin dependent). Currently, d iabetes is recognized as the world’s most common metabolic disorder, affecting people globally and of all age groups. For the year 2000, it was estimated that over 175 million people worldwide, were affl icted with this disease (International Diabetes Institute, World Health Organisation). Clinically, diabetes is primarily characterized by fasting hyperglycemia, is often associated with cardio- vascular risk factors, and may lead to severe complications. At present, type I diabetes comprises about 5–10% of all diagnosed cases. The molecular complexity of this disorder is well documented, and current therapies revolve around exogenous insulin supplementation [1,2]. On the other hand, type II diabetes accounts for the remaining 90–95% of the cases. At the molecular level, a postreceptor defe ct of insulin signaling is mainly thought to und erlie the basis of insulin resistance in type II diabetes [3]. Consequently, understand- ing the mechanisms by which this may occur will provide invaluable insight for the development of novel therapies. In the present review, we will summarize the current under- standing of insulin signaling with particular focus on how protein tyrosine phosphatases regulate this process. BRIEF OVERVIEW OF INSULIN SIGNALING Insulin is a pleiotropic hormone with multiple integrated signaling pathways. For brevity, we will only describe those relevant to this review. The insulin receptor (IR) belongs to a subclass of the large family of protein tyrosine kinases [4]. It is a t ransmembrane protein c omprising two extracellular a subu nits and two tran smembrane b subunits (Fig. 1). Upon binding to insulin, the intrinsic kinase activity of the receptor is increased, and the IR undergoes autophospho- rylation on several tyrosine residues located on the cyto- plasmic portion of the b subunits [5]. Subsequently, these phosphotyrosine residues, in their surrounding seq uence Correspondence to M. L. Tremblay, Department of Biochemistry and McGill Cancer Center, McGill University, 3655 Promenade Sir William Osler, Room 715, Montreal, Quebec, Canada, H 3G 1Y6. Fax: + 1 514 398 6769, Tel.: + 1 514 398 7290, E-mail: tremblay@med.mcgill.ca Abbreviations: GLUT4, g lucose transporter 4; IR, insulin receptor; IRS, insulin receptor substrate; PTP, protein tyrosine phosphatase; SH2, Src homology 2; PI3-kinase, phosphatidyl inositol 3-kinase. (Received 6 August 2001, accepted 20 September 2001) Eur. J. Biochem. 269, 1050–1059 (2002) Ó FEBS 2002 context, recruit signaling m olecules containin g SH2 ( Src homology 2) or PTB (phosphotyrosine binding) domains [6]. Although not limited to, most of the recruited proteins belong to a class of adapte r p roteins of the insulin receptor substrate (IRS) family [7,8]. The best characterized examples include Shc, which is primarily involved in the activation of the mitogen activated protein kinase (MAPK) pathway for mitogenic effect; and IRS-1, which can transmit insulin signaling to both metabolic and mitogenic processes [9,10]. The primary function of insulin is to maintain glucose homeostasis. For most ce lls, this is ach ieved via the insulin dependent translocation of the glucose transporter, GLUT4, from intracellular vesicles to the cell surface [11– 13]. Upon recruitment of IRS-1 to the activated IR, IRS-1 becomes heavily tyrosine phosphorylated and serves as a large scaffolding protein by binding to several SH2 containing proteins. The most prominent e xample is the p85 regulatory s ubunit of phosphatidylinositol 3-kinase (PI3-kinase). Binding to p85 recruits the p110 catalytic subunit of PI3-kinase, resulting in the activation of the PI3- kinase pathway, a necessary component involved in GLUT4 translocation. However, activation of PI3-kinase alone is insufficient, as platelet-derived growth factor which also activates PI3-kinase, does not promote glucose trans- port [14,15]. In f act, recent studies in 3T3-L1 adipocytes have demonstrated a pathway parallel to PI3-kinase, which is required for insulin-mediated G LUT4 translocation. Activation of the IR results in the formation of a protein complex involving the scaffolding proteins CAP and Cbl. Subsequent tyrosine phosphorylation of C bl by the IR leads to translocation of the Cbl–CAP complex to lipid rafts, a process that is necessary for GLUT4 translocation [14,15]. In contrast to the activation and propagation of insulin signal transduction, the negative regulatory components that attenuate insulin signaling are less well defined. Because tyrosine phosphorylation o f t he IR corr elates with its activity and function, the protein tyrosine phosphatases [16] are prominent candidates t o negatively regulate i nsulin action. Indeed, vanadium compounds, known inhibitors of PTPs, have long been known t o possess insulin mimetic or enhancing effects [17–19]. However, it should also be noted that there is evidence f or serine phosphorylation [5] and O-glycosylation [20] in attenuating insulin signaling, but these will not be discussed here. THE PROTEIN TYROSINE PHOSPHATASE FAMILY PTPs represent a large family of enz ymes t hat rival the PTKs in both functional and structural diversity. Members of this group can b e classified into receptor vs. nonreceptor PTPs. Common to all members is a highly conserved core of about 250 amino acids that make up the catalytic domain. The PTP signature motif, V/IHCSAGXGRXG sequence contains an invariant cysteine residue that is critical for PTP activity [21]. In addition, s ome receptor PTPs (rPTPs) possess two such d omains, although only one is usually active [22–24]. Apart from the catalytic domain, the rest of the protein is quite divergent amongst PTPs. For the sake of simplicity, and to illustrate the point, we will only depict several PTPs relevant to insulin signaling (Fig. 2). A s ubset of rPTPs contain structural motifs such as immunoglobulin-like and fibronectin type III elem ents. These structures have been found in cell-adhesion molecules and suggest a role for these PTPs in cell–cell contact or cell– extracellular matrix interactions. On the other h and, all intracellular PTPs possess a single conserved phosphatase domain, that is flanked at either the N- or C-terminus by noncatalytic segments. T hese segments play regulatory roles, either by binding each PTP to its substrate(s) or to adapter molecules through domains that regulate protein– protein interactions, by targeting the PTP to a particular subcellular compartment, or by keeping the enzyme in an inactive conformation. To elucidate the function of PTPs and t heir mechanisms of action, identification of their substrates is critical. Over the past decade, many studies on PTPs have utilized a strategy called Ôsubstrate trappingÕ [25]. In this method, mutagenesis of the conserved cysteine to serine (CfiS) or an aspartate to alanine (DfiA) within the catalytic domain of Fig. 1. Scheme of the major insulin signaling pathways. The activated insulin receptor phosphorylates tyrosine residu es on IRS p ro- teins, Shc, CAP and other intracellular sub- strates. These substrates then bind to various downstream signaling effectors, transmitting the metabolic and mitogenic signal of insulin. CAP, c-Cbl-associating protein; FRAP/ mTOR, mammalian target of rapamycin; MAP, mitogen activa ted protein ; MAPK, MAP kinase, MEK, MAP/ERK kinase; PI3- kinase, phosphatidylinositol 3-kinase; PKB/ Akt, protein kinase B; SHP-2, SH2 containing phosphatase-2; Sos, Son of sevenless. Refer to text for more details. Ó FEBS 2002 Protein tyrosine phosphatases in insulin signaling (Eur. J. Biochem. 269) 1051 PTPs eliminates their e nzymatic ac tivity. However the resulting mutated enzymes are still able to recognize their specific targets with complete loss of or reduced ability to catalyze the removal of the phosphate moiety from tyrosine. Thus, these Ôtrapping mutantsÕ provide a convenient means to isolate potential substrates of PTPs in a rapid and efficient way. In a modified approach, this trapping strategy was used in combination with gene targeting technology to identify physiological substrates of PTPs [26]. CANDIDATE IR PTPs As a first step to identify PTP s that play a regulatory role in insulin signaling pathway, several groups studied the expression profile of PTPs expressed in the major insulin sensitive t issues. For example, rPTPa,LAR,SHP-2and PTP-1B have been identified as the four major PTPs in rat adipocytes [27]. Moreover, immunodepletion studies in rat skeletal muscle demonstrated that LAR, SHP-2 and PTP- 1B were the t hree major enzymes responsible for PTP activity [28]. Further compelling eviden ce for these PTPs in insulin signaling stems from the f act t hat the expression levels and/or activity these specific PTPs are increased in insulin resistant obese patients [29]. LAR LAR belongs t o a subfami ly of r PTPs that also include PTPr and PTPd. Members o f this subfamily are expressed as preproteins and undergo proteolytic processing to generate a molecule containing two cytoplasmic c atalytic domains linked through a single hydrophobic transmem- brane stretch to a large extracellular segment (Fig. 2). An additional proteolytic cleavage site near the transmembrane stretch a llows shedding of the extracellular domain, and has been s uggested to be a mechanism c ontrolling LAR function [30,31]. The extracellular segment consists of three immunoglobulin-like r epeats and four to eight type-III fibronectin repeats. On the cell surface the two subunits of LAR form a complex of two noncovalently associated subunits [32,33]. The localization of this rPTP makes it a logical candidate for d ephosphorylation of the IR. Indeed, an a ssociation between LAR and the I R h as been demonstrated by coimmunoprecipitaition studies in cells [34]. Furthermore, these studies also showed that insulin treatment increased the amount of IR/LAR complex detected. Consistent with these results, overexpression or antisense suppression stud- ies of LAR sh owed that this rPTP could negatively regulate IR, IRS-1 and Shc phosphorylation, as wel l as the P I3- kinase and M APK pathways [35– 37]. In C HO-hIR cells expression of LAR reduced insulin stimulated tyrosine phosphorylation of IR and IRS-1, as well as DNA synthesis [38]. Importantly, proper membrane localization of LAR seems to be required, as expression of the cytoplasmic domain of LAR alone does not recapitulate these effects. Studies with knockout mice indicate that, although L AR is not required for embryonic development, it seems to be necessary for mammary gland development [39]. The effect of LAR deficiency on insulin signaling has yet t o be reported in these mice. Using a different strategy, Skarnes et al. g enerated transgenic mice expressing reduced (near undetectable) levels of LAR transcript [40,41]. Studies in this model were performed to provid e some in vivo evid ence that LAR is involved in glucose homeostasis and insulin signaling [42]. However, insulin stimulated receptor phos- phorylation and basal PI3-kinase activity were only modestly increased under reduced LAR expression. Further- more, I RS-1 tyrosine phosphorylation was unaffected. In contrast, insulin stimulated PI3-kinase activity was diminished in these mice compared to controls. It should be noted that the importance of LAR for p roper neuronal development [41,43] makes the situation a complex one. To overcome the phys iological complexity of LAR, transgenic mice overexpressing this rPTP in skeletal muscle (MCK-hLAR mice) were developed [44]. Importantly, this model was intended to approximate the increased expres- sion of LAR in insulin-resistant humans. MCK-hLAR mice maintain glucose levels at higher plasma insulin levels, and glucose uptake is reduced in skeletal muscles, compared to controls. In muscle tissue of these mice, insulin induced IR and IRS-1 phosphorylation is normal, but IRS-2 phos- phorylation is decreased. Although IRS-1 can be dephos- phorylated by LAR in vitro [45], studies on IRS-2 have yet to be performed. Furthermore, IRS-1 o r IRS-2 associated PI3-kinase activity was also diminished. Taken together, these results suggest that LAR negatively regulates insulin signaling primarily through d ephosphorylation of IRS-2 (or other IRS proteins), although IR and IRS-1 may be affected in other tissues or physiological states. Finally, MCK- Fig. 2. The prominent protein tyrosine phos- phatases implicated in insulin signal transduc- tion. Structure of several phosph atases implicated in insulin signal transduction. rPTP-a, LAR, P TP-1B an d SHP-2 are the major phosphatases acting on the insulin signaling pathw ay. rPTP-e, r PTP-r and TC-PTP are candidates shown by in vitro binding and dephosp horylation a ssays or suggested by their structure similarity to phosphatases involved in the insulin signaling path way. 1052 A. Cheng et al. (Eur. J. Biochem. 269) Ó FEBS 2002 hLAR mice also provide an important model t o understand the role of LAR in the pathogenesis of insulin resistance. RPTPa rPTPa mRNA is expressed in m ost tissues, w ith highest expression in brain and kidney, suggesting that this PTP could play a fundamental role in the physiology of all cells [46]. The best-characterized substrate of rPTPa is the Src kinase, with particular emphasis in the context of transfor- mation and neuronal differentiation [47,48]. In addition, p130Cas [49] and the IR [50] have also been shown to be potential substrates of rPTPa.ExpressionofrPTPa in cells can inhibit some of insulin mediated effects. For example, expression of rPTPa in BHK-IR cells inhibits insulin- mediated cell rounding and growth inhibition of those cells, concomitant with increased IR phosphorylation [51]. Fur- thermore, in rat adipocytes, rPTPa decreases in sulin stimu- lated GLUT4 cell surface translocation [ 52]. In contrast, antisense studies in 3T3-L1 adipocytes showed that rPTPa was dispensable for insulin induced MAPK activation and DNA synthesis [53]. Although mice deficient in rPTPa have demonstrated the importance of this P TP in the activation of Src kinases [54,55], its physiological importance in insulin signaling remains unclear. SHP-2 SHP-2 [56] is a widely expressed nonreceptor PTP that contains two N-terminal SH2 domains, a C-terminal catalytic domain and a C-terminal s egment containing two tyrosyl phosphorylation sites (Fig. 2). The SH2 domains of SHP-2 bind many a ctivated growth factor receptors as well as IRS-1 [57–59]. It has been suggested that these associations displace intramolecular i nteractions of SHP-2, leading to a conformationally more open state and increased catalytic activity [6 0–62]. In contrast to many other growth factor receptor associated PTPs, S HP-2 does not seem to dephosphorylate the receptor. In fact, genetic studies indicate that SHP-2 is a positive effector of growth factor receptor signaling [63]. However, Kuhne et al. [64] proposed that the binding of IRS-1 to SHP-2 enhances its phosphatase activity toward IRS-1, resulting in its dep- hosphorylation in vivo. Thus SHP-2, in contrast with most other PTPs, may a ct as ei ther a positive o r n egative regulator of growth f actor signaling. Many studies suggest that SHP-2 binds to both the IR and IRS-1. Forexample, t he IR and SHP-2 interact in ayeast t wo- hybrid assay [65]. Transfection experiments demonstrated that this association is mediated be tween the proximal SH2 domain of SHP-2 and phosphotyrosine 1146 of the activated insulin receptor [66]. Insulin also induces the formation of a complex of IRS-1 and SHP-2, requiring the tyrosines 1172 and 1222 of IRS-1 [59,67]. However, others have suggested that SHP-2 is not the major protein complexed with IRS-1 in insulin stimulated 3T3-L1 adipocytes [68]. Microinjection of in terfering molecules [69], overexpres- sion of dominant negative mutants [70–72], a nd genetic studies [63] indicate that SHP-2 is required for activation of the MAPK p athway by a variety of growth factors, including insulin. However, the requirement for SHP-2 binding to IRS-1 for this pathway is unclear [69,73]. SHP-2 canalsobindtoIRS-2,IRS-3[74]andIRS-4,suggesting possible functional redundancy for the SHP-2/IRS-1 association. In addition, SHP-2 binding to SHPS-1 [SH2- domain bearing protein tyrosine phosphatase (SHP) sub- strate-1] [75] may provide an additional pathway for insulin induced MAPK activation. In ins ulin-induced metabolic signaling, a SHP-2 C-S mutant slightly impaired GLUT4 translocation in primary adipocytes, whereas wild-type SHP-2 did not [76]. Genetic studies in mice indicate that SHP-2 is required for embryonic d evelopment [ 77,78]. S HP-2 heterozygous knockout mice are v iable, and in these mice, plasma insulin and glucose uptake were normal [78]. Moreover tyrosine phosphorylation of IR and IRS-1 from muscle tissue was similar to that of wild-type controls. These results suggest that SHP-2 may play a minor role in the metabolic effects of insulin that may not be detectable unless SHP-2 function is completely removed. Perhaps tissue specific knockouts of SHP-2 can further address the issue. In another approach, t he transgenic expression of a mutant SHP-2 was studied [79]. This mutant (DeltaPTP) contains the two SH2 domains but lacks the PTP domain and the C-terminal tyrosines. In DeltaPTP mice, insulin induced association of endogenous SHP-2 with IRS-1 was reduced, suggesting a dominant negative effect of the mutant SHP-2 protein. Furthermore, DeltaPTP mice are insulin resistant, and i nsulin-mediated t yrosine phosphorylation of IRS-1, stimulation of PI3-kinase and Akt activities were attenuated in muscle and liver. T hus, t he inhibition of endogenous SHP-2 by these dominant negative studies suggests a positive role for SHP-2 in insulin-induced metabolic signaling. Because DeltaPTP mice are viable, it suggests that the SH2 domains of SHP-2 a lone, are able to mediate a spects of signaling required for embryonic development. PTP-1B PTP-1B was the first mammalian PTP identified and purified to homogeneity [80]. This phosphatase is widely expressed and localizes predominantly to the ER through a cleavable proline-rich C-terminal segment (Fig. 2) [81,82]. Moreover, the C-terminal 35 amino acids of PTP-1B were found both necessary and sufficient for its targeting to the ER [81]. Cleavage of this segment appears to release the enzyme from the ER and increase its specific activity [83]. By in situ hybridization, Brown-Shimer et al. [84] identified PTP-1B as a single-copy gene that mapped to the long arm of human chromosome 20 in the r egion q13.1–q13.2. Interestingly this region was identified as a quantitative trait locus linked to obesity and insulin [85]. Studies using the CfiSmutantofPTP-1B(PTP-1B C215S) have demonstrated an association of PTP-1B with the IR [86,87]. Upon insulin treatment, PTP-1B becomes tyrosine phosphorylated at three sites (Tyr 66, 152, 153), and mutation of any of these residues impairs its association with the activated IR [87]. Within the IR, the binding occurs in a region containing residues 1142–1153 [88], and muta- tion of tyrosines 114 6, 1150, and 1151 diminish the association [ 87,89]. Indeed, crystal structure and kinetic studies provide evidence t hat P TP-1B preferentially dep- hosphorylates tyrosines 1150 a nd 1151 of the IR [90]. In addition to the IR, IRS-1 might also be a substrate of PTP-1B [45]. Furthermore, in the presence of Grb2, IRS-1 dephosphorylation by PTP-1B is accelerated. Thus, these Ó FEBS 2002 Protein tyrosine phosphatases in insulin signaling (Eur. J. Biochem. 269) 1053 results suggest that PTP-1B could negatively regu late insulin signaling by acting on t wo different componen ts of the pathway. A plethora of s tudies demonstrate that PTP-1B can attenuate insulin signaling. Microinjection of homogeneous preparations of PTP-1B protein into Xenopus oo cytes decreases t yrosine phosphorylation of proteins correspond- ing to the molecular m ass of the IR. Correspondingly, insulin-induced S6 kinase activity and meiotic cell division were retarded as well [91,92]. In mammalian cells, osmotic loading of PTP-1B antibodies decreases insulin induced IRS-1 phosphorylation, PI3-kinase activity, as well as DNA synthesis [93]. Finally, overexpression of PTP-1B reduces glucose uptake and GLUT4 translocation to the cell membra ne [ 76, 94]. Regulation of insulin s ignaling b y PTP-1B appears to be tissue specific. Overexpression of PTP-1B in 3T3-L1 adipo- cytes attenuates insulin induced IR, IRS-1 phosphorylation, as well as PI3-kinase and MAPK activation [95]. However, neither Akt activation nor glucose transport seemed to be affected. Thus, it i s possible that PTP-1B may regulate insulin-mediated mitogenic, as opposed to metabolic events in this cell type. In contrast, overexpression of PTP-1B in L6 myocytes and Fao hepatoma cells attenuated insulin- induced Akt activation and glycogen synthesis [96]. An increasing amount of evidence suggests that insulin signaling can inhibit PTP-1B activity, perhaps as part of a negative feedback loop. For example, insulin stimulation of 3T3-L1 adipocytes induces a burst of intracellular hydrogen peroxide that is thought to reversibly oxidize an d t hus inactivate the invariant cysteine in the catalytic domain of PTP-1B [97]. In another study, it was suggested that insulin could also down-regulate PTP-1B activity by suppressing serine phosphorylation and activation on the phosphatase via an unidentified mechanism [98]. Knockout studies in mice provided in vivo confirmation that PTP-1B is a bona fide phosphatase of the IR [99,100]. Despite its involvement in a variety of signaling p rocesses, PTP-1B is surprisingly not required for embryonic devel- opment, and PTP-1B-deficient mice grow and d evelop normally with similar lifespans to wild-type littermates. However, PTP-1B-deficient mice display increased insulin induced IR phosphorylation i n liver and muscle but n ot adipose tissue. IRS-1 phosphorylation was also increased in muscle, but it is unclear whether this is because IRS-1 is a substrate of PTP-1B, or an increased IR activity in knockout mice. Furthermore, PTP-1B-deficient mice are hypersensitive as assayed b y oral g lucose tolerance t ests, intraperitoneal insulin tolerance tests, and blood levels of glucose and insulin. Importantly, PTP-1B-deficient mice remained insulin sensitive when f ed a high fat diet. Strikingly, though, they were also resistant to obesity, due in part to a decrease in fat cell mass and increased energy expenditure. These results suggest that PTP-1B is a major modulator of insulin sensitivity and fuel metabolism, and point to PTP-1B as a potential therapeutic target f or the treatment of type II diabetes and obesity. Importantly the insulin receptor phosphorylation appears to be modified in liver and muscle tissues but not in adipose tissue, suggesting that although PTP-1B is a major modulator of the IR, other PTPs may have tissue specific preferences for the insulin receptor, in particular in adipocytes. OTHER CANDIDATE PTPs RPTPr Evidence for a functional r ole of rPTPr in insulin signaling has not been reported to date. However, its similarity with LAR, and the fact that it is expressed i n relatively high levels in insulin sensitive tissues (higher than LAR) [101] make rPTPr a possible c andid ate to regulate insulin signaling. Genetic studies wit h rPTPr deficient mice reveal the presence of primarily neuroendocrine defects [102,103]. However, preliminary s tudies also indicate that rPTP r deficiency leads to insulin hypersensitivity f rom measure- ments of fasting glucose and insulin levels (X. Elchebly & M. L. Tremblay, unpublished observations). As in the case with the LAR knockout it cannot be ruled out that the effects on insulin signaling are secondary to the neuroendo- crine status. Thus generation of transgenic lines over- expressing this PTP or the creation of tissue specific knockouts should answer this question. rPTPe rPTPe is similar in structure to rPTPa. In addition to rPTPa, expression of rPTP e in BHK-IR cells also inhibits insulin mediated cell rounding and g rowth in hibition o f BHK-IR cells, and requires membrane localization of the PTP [51,104]. TC-PTP, rPTPd and Sap-1 In an attem pt to f urther extend the list of c andidate IR PTPs, a mass screen approach utilizing in vitro binding and dephosphorylation a ssays were performed on a large list o f PTPs [105]. In this study, in addition to PTP-1B, three other PTPs were suggested to be important for IR dephosphory- lation: TC-PTP, rPTPd and Sap-1. Although not all PTPs tested performed well in these assays, one must consider that for each PTP, their physiological situation is unique and several other factors are implicated. For example, these may include: t issue distribution, subcellular localization, as well as the significance of additional binding partners to form functional multiprotein complexes. COMPARTMENTALIZATION OF IR AND PTPs An increasing amount of evidence suggests that regulation of insulin signaling by PTPs may also occur at the level of compartmentalization. In the absence of ligand, the I R normally resides a t the plas ma membrane. U pon insulin binding, the ligand–receptor complex is rapidly sequestered from the p lasma membrane a nd internalized into endo- somes within several minutes [106,107]. Here, the acidic pH of endosomes induces the dissociation of insulin from IR and allows the degradation of insulin by endosomal acidic insulinase [108]. The IR is then recycled back to the cell surface. However, under conditions of prolonged stimula- tion with saturating levels of insulin, a subset of the IRs are transported t o the late endosome a nd lysosome for degra- dation [109,110]. Although the IR kinase activity is required for ligand- stimulated IR internalization, the role of IR internalization 1054 A. Cheng et al. (Eur. J. Biochem. 269) Ó FEBS 2002 on insulin receptor signaling remains unclear. The endoso- mally associated IRs h ave been reported to exhibit a transient elevation of the tyrosine phosphorylation and to achieve the full activation of the receptor itself as well as th e activation of IRS-1 and PI3-kinase (reviewed in [111]). In contrast, studies using a dominant negative dynamin molecule that blocks IR internalization showed that the inhibition o f IR endocytosis had no major effect on IR autophosphorylation and IRS-1 tyrosine phosphorylation [112]. Even though a 50% decrease in the insulin activated PI3-kinase activity was observed when IR internalization is blocked, it did not affect the subsequent Akt phosphory- lation and activation. The only major defect caused by inhibition of IR internalization was impaired Shc tyrosine phosphorylation and MAPK activation. In summary, most of the acute actions of insulin could be initiated by activation of the plasma me mbrane-localized insulin receptor. While the activation of the insulin signaling cascade appears to be independent of IR internalization, tyrosine phosphatase activity towards IR is l argely observed in endosomes (reviewed in [111,113]). Studies in rat hepatoma cells demonstrated that internalized IRs w ere dephosphory- lated and inactivated prior t o recycling back to the plasma membrane [114]. Using isolated rat liver endosomes, Faure et al. sh owed that a substantial amount of IR PTP activities is tightly associated with the endosomes. This activity resists the 0.6 M KCl treatment of the endosomal membrane, but Triton X-100 totally abolishes dephosphorylation [115,116]. These studies strongly suggest that t he endosome is a major site of IR dephosphorylation. However, due to the large number of phosphotyrosine residues o n the IR, and the complexity of insulin signaling, the plasma membrane localized IR should not be discarded as an important site of PTP action. For rPTPs such as LAR and rPTPa, the plasma membrane is the obvious location for IR dephosphorylation. As previously discussed, membrane targeting of these rPTPs seems to be necessary for IR dephosphorylation. Yet, LAR has also been detected i n r at hepati c e ndosomes [34]. Although the kinetics of LAR internalization upon insulin administration was much slower ( 30 min), compared to that of IR ( 2–5 min), incubation of endosomal fractions with antibodies against LAR reduced IR dephosphorylation by about 28%. In addition, subcellular fractionation of rat adipocytes showed that both LAR and rPTPa are present in heavy microsomes [117]. Although the identity of these h eavy microsomes was not determined, the presence of increased IR in the same fraction after insulin stimulation suggests that these membranes likely contain endosomal compartments. Amongst the intracellular P TPs, PTP-1B is a c lear physiological regulator of the IR, and perhaps IRS proteins as well. Although a truncated form of PTP-1B was initially identified in the cytosolic fraction of human placenta, the subcellular characterization of full length PTP-1B demon- strated its predominan t localization in the ER through an association with its C-terminal 35 amino acids [81,82]. Other Fig. 3. Model of coordinated PTP action on the insulin signaling pathway. Different PTPs may act on the IR in various compartments within the cell. For example, the transm embrane phosphatases LAR and rPTP-a may p redomin antly act at the plasma membrane on either the receptor or downstream substrates. On the other hand, PTP-1B could act on IR and IRS-1 at the plasma membrane and/or endosomes. Finally, the cytosolic PTP, SHP-2, could potentially be re cruited to man y sites of insu lin action. Howeve r, the role of SHP-2 is m ainly to transmit positive s ignals from the IR. How these PTPs coordinate their ac tion on the insulin signaling pathway remains to be determined. Ó FEBS 2002 Protein tyrosine phosphatases in insulin signaling (Eur. J. Biochem. 269) 1055 reports indicated that s ubstantial a mounts o f full length PTP-1B are also found in the cytosol of rat fibroblasts and skeletal muscle [28]. In rat adipocytes, PTP-1B was found in the light microsome f raction a nd to a l esser extent, the cytosol and heavy m icrosomes [117]. H owever, immuno- blotting failed to reveal th e presence of PTP-1B in rat liver endosomes [116]. Thus PTP-1B could dephosphorylate the IR in the light and heavy microsomes, or cytosolic PTP-1B may be recruited to th e appropriate site. The precise localization where PTP-1B dephosphorylates the IR and the mechanism of P TP-1B translocation to t he site o f IR dephosphorylation remain to be elucidated. Finally, in r esponse to i nsulin, s ignificant amounts o f IRS-1 and IRS-2 a re also associated with internal mem- branes in rat a dipocytes [1 17–119]. Furthermore, i nsulin stimulation in rat liver increases the association of active IRS-1, IRS-2, and PI3-kinase to endosomal fractions [120]. These d ata further show that there is a complex spatial control in insulin receptor signaling of t he various molecules that are i nvolved a nd support an important role of the subcellular localization of both the tyrosine kinases, their substrates and the PTPs involved in insulin signaling. CONCLUSIONS AND PROSPECTS In contrast to what we have depicted in Fig. 1, the metabolic and mitogenic pathways emanating from the IR are diverse and complex [121]. Although not limited to this, the action of PTPs represents an important aspect in both the transmission and attenuation of insulin signaling. Indeed, many s tudies have been aimed at developing inhibitors towards these PTPs that might c ircumvent insulin resistance and treat type II diabetes. Currently, both PTP- 1B and L AR are s trong candidates for inhibitor design studies, although PTP-1B has been the major focus due to its s maller s ize, the remarkable d ata from the knockout mice, and the availability of structural and kinetic data. An emerging theme that requires further study is how the coordinate actions of several PTPs may regulate insulin signaling (Fig. 3). As a cytosolic protein, SHP-2 could potentially be recruited t o many sites of insulin action and positively participate in signal transduction, either through direct binding with the IR, or through adapter molecules such as IRS proteins. On the other hand, the rPTPs probably dephosphorylate the IR (or I RS proteins) a t the plas ma membrane, a lthough evidence s uggests other subcellular compartments are a possibility as well. For PTP-1B, several sites of insulin action seem possible. It will be interesting to determine how different PTPs m ight temporally, as well as spatially, regulate insulin signaling under normal physiolo- gical conditions and in pathophysiological states such as diabetes. Finally, it still remains t o be determined whether different PTPs may act on specific phosphotyro sine residues on the IR, thus providing another level of specificity. ACKNOWLEDGEMENTS We wish to thank John Wagner for critical reading of the manuscript and helpful discussions. A. C. is a recipient of a Medical Research Council studentship. N. D. is a recipient of a Canadian Institutes of Health Research doctoral award. F. G. is a recipient of a Human Frontiers postdoctoral fellowship. M. L. 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PTPs coordinate their ac tion on the insulin signaling pathway remains to be determined. Ó FEBS 2002 Protein tyrosine phosphatases in insulin signaling (Eur.