Role of Protein Tyrosine Phosphatases in Growth Factor and Cytokine Signaling

The binding of growth factors, cytokines, and certain hormones to their cell surface receptors results in the rapid activation of PTKs. These initial tyrosine phosphorylation events result in diverse changes in cell physiology and metabolism, cell growth and differentiation, and cell motility and shape. The receptors for many growth factors and certain hormones have intrinsic tyrosine kinase activity within their intracel-lular domains. Ligand-induced oligomerization activates these receptor tyrosine kinases (RTKs) and facilitates transphosphorylation of multiple Tyr residues within their cytoplasmic domains. These phos-photyrosine residues serve as sites for the docking of adaptor molecules that can activate diverse downstream signaling mechanisms. Although devoid of intrinsic kinase activity, cytokine receptors trigger tyrosine phosphorylation of their cytoplasmic domains through the action of associated JAK family kinases that are activated when ligands trigger receptor oligomerization. The signals emanating from both the growth factor RTKs or cytokine receptors are propagated by signaling pathways, most of which include further activation of downstream protein kinases such as those of the MAP kinase cascade.

Many CX5R phosphatases have been implicated in regulating mitogenic signaling both at the level of the receptor and further downstream. Much of the evidence linking PTPs to these signaling pathways remains indirect and circumstantial, primarily because of a lack of knowledge regarding substrates. In this section, we focus on the tyrosine-specific enzyme SHP-2 and a subset of the DSPs known as the MAP kinase phosphatases (MKPs). Both types of phospha-tases have been intensively studied and they will serve to illustrate how enzymes of the PTP family regulate mitogenic signal transduction.

6.3.1. SHP-2/Csw Function Positively in Mitogenic Signaling

Both genetic and biochemical studies have shown that SHP-2 from vertebrates and its orthologs encoded by the corkscrew (csw) and ptp-2 genes from Dro-sophila and C. elegans, respectively, are required for signaling through multiple RTKs and cytokine receptors. SHP-2 has been shown to function downstream of receptors for epidermal growth factor (EGF), insulin, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and numerous cytokines such as IL-3, IL-2, IL-5, and growth hormone.

Many developmental processes in both vertebrate and invertebrate organisms depend on RTK signaling pathways. In genetic studies, Csw was identified first as a component of the Torso RTK signaling pathway that determines cell fate in terminal regions of the Drosophila embryo. Subsequent studies in Drosoph-ila revealed a role for the Csw phosphatase in multiple RTK signaling systems including differentiation of the R7 photoreceptor of the ommatidium in the compound Drosophila eye, which is controlled through the Sevenless (Sev) RTK. The C. elegans ptp2 phos-phatase is essential for oogenesis and also functions downstream of the EGF receptor homolog. Mice, in which the SH2 domains of SHP-2 were deleted, exhibited serious developmental defects. It has been suggested that SHP-2/Csw and their orthologs may function in most if not all RTK signaling pathways.

The phosphatase activity of SHP-2, Csw, and their orthologs is required for their positive role in promoting RTK and cytokine signal transduction. With their ability to oppose protein tyrosine kinases, the positive role for SHP-2/Csw phosphatases in these pathways might appear paradoxical. Although many of the molecular details are not yet understood, there appear to be at least three distinct mechanisms whereby SHP-2/Csw carry out their positive function in mitogenic signal transduction. SHP-2 and Csw function downstream of the receptors but act both upstream and in parallel to the Ras/MAP kinase cascade (see below) that mediates many of the effects of growth factors and cytokines.

Signals arising from RTKs, cytokine receptors, and many other cell surface receptors are propagated through one or more MAP kinase cascades that deliver signals to the nucleus to trigger gene expression and cell proliferation as shown in Fig. 10. There are many MAP kinase signaling modules but they all are comprised of three protein kinases that act on one another in series. The terminal protein kinase in the series is the MAP kinase (MAPK), which is a Ser/Thr protein kinase that is activated when phosphorylated on Thr and Tyr residues within the "activation loop" of the kinase catalytic domain. MAPK is downstream of and phosphorylated by the dual specificity protein kinase, MAP kinase kinase (MAPKK), which is, in turn, activated upon phosphorylation by a MAP kinase kinase kinase (MAPKKK). Raf kinase functions as a MAPKKK that is linked to RTKs and many other receptors through the Ras GTP binding protein. Ras is regulated by the guanine nucleotide exchange factor known as Sos. When the cytoplasmic domain of an RTK is phosphorylated in response to ligand, the SH2 domain containing adaptor protein Grb2 translocates to the receptor carrying its binding partner Sos. Once recruited near the plasma membrane via the interaction of Grb2 with an RTK, Sos stimulates the formation of an activated Ras-GTP complex, which in turn activates the Raf kinase and initiates the MAP kinase cascade (see Fig. 10).

A signaling mechanism for Csw and SHP-2 has been identified in studies of the Drosophila Torso RTK and several mammalian growth factor receptors such as the PDGF receptor (see Fig. 10). Ligand binding and the subsequent tyrosine phosphorylation of Torso provide a docking site for the recruitment of Csw via its SH2 domain. On association with Torso, Csw is phosphorylated on a Tyr residue located near its C-terminus. Once this site is phosphorylated, Csw binds the SH2 domain of Drk (the Drosophila homolog of Grb2). Thus, Drk and its constitutively associated partner Sos are linked to the Torso RTK through Csw. When translocated to a site on Torso near the membrane, Sos activates Ras and triggers the MAP kinase cascade as outlined previously. Acting in this capacity, Csw is not only a phosphatase, but also

Fig. 10. Role of protein tyrosine phosphatases in mitogenic signaling. Three major roles of SHP-2 in mediating signaling from RTKs are illustrated in this generalized scheme. SHP-2 is recruited to RTKs when ligand binding leads to autophos-phorylation of Tyr residues recognized by the SH2 domains SHP-2. Other adaptor proteins and signaling enzymes that are typically recruited to RTKs are not shown. When SHP-2 is recruited to the RTK, it is phosphorylated on a C-terminal Tyr, creating a binding site for SH2 domains of Grb2. SHP-2 then couples Grb2 to the activated RTK, thus triggering the "classic MAP kinase" signaling cascade as shown. SHP-2 is also recruited to the Gab2 adaptor protein, which may be its substrate. The association of SHP-2 and Gab2 activates gene expression in a pathway that acts in parallel with the MAP kinase cascade as shown. Finally, SHP-2 is recruited to SHPS-1 and other members of a family of membrane proteins that are phosphorylated upon growth factor activation. Recruitment of SHP-2 to this complex may lead to MAP kinase activation but this mechanism is not well understood. The role of MKP1 in negatively regulating the MAP kinase cascade is also illustrated. As shown, MKP1 opposes the MAP kinase kinase that phosphorylates and activates MAP kinase. This scheme represents a generic model for three positive roles of SHP-2 in mitogenic signaling, but it is not meant to suggest that all three modes of signaling are used by a single receptor or that all three modes would operate simultaneously in a single cell.

an adaptor molecule linking other signaling proteins to Torso. When Csw is bound to Torso, its phospha-tase activity is stimulated on engagement of its SH2 domain and it dephosphorylates a Tyr residue within the cytoplasmic domain of the receptor. In the phos-phorylated state, this Tyr residue serves as a site for binding Ras-GAP, which is a negative regulator of Ras. Thus, the displacement of Ras-GAP from Torso by Csw also promotes Ras activation. With certain mammalian growth factor receptors, SHP-2 also acts as an adaptor protein linking RTKs to Grb2 and Sos, which then leads to activation of the Ras-MAP kinase cascade. It is not known whether SHP-2 also dephos-phorylates and regulates RTKs in a manner analogous to Csw.

Csw also binds and dephosphorylates the Drosoph-ila adaptor protein Dos that is required for signaling by Sev and other RTKs with developmental roles. Dos has an N-terminal pleckstrin homology (PH) domain with sequence similarity to a segment of Gabl, IRS-1, and IRS-2, which are mammalian growth factor receptor substrates. Moreover, the C-terminus of Dos has a polyproline region and tyrosines that are potential binding sites for SH3 and SH2 domains. Genetic analyses indicate that Csw and Dos either act in parallel or upstream of Ras in Sev signaling.

A recent study has identified Gab2 as an important adaptor protein required for signaling downstream of cytokine and RTK receptors (see Fig. 10). Upon cytokine or growth factor stimulation, Gab2 is phos-phorylated and binds SHP-2 as well as other signaling proteins such as the adaptor protein Shc or the p85 subunit of phosphoinositide-3 kinase (PI-3 kinase). The available evidence suggests that Gab2 is a substrate of SHP-2. As mentioned previously, Gab2 and Dos have limited sequence similarity and related structural organization. The Gab2/SHP2 complex is thought to activate cytokineinduced gene expression in a pathway that is parallel to the Ras-MAP kinase cascade (see Fig. 10). These studies suggest that SHP-2 and Csw have a second, conserved mode of action in mitogenic signaling in which they act together with the adaptor proteins Grb2 and Dos, respectively, to propagate signals that activate gene expression.

SHP-2 may function through yet a third mechanism in growth factor and cytokine signaling (see Fig. 10). In response to insulin and other mitogens, a transmembrane glycoprotein known as SHPS-1 is phos-phorylated permitting the subsequent recruitment of SHP-2. Although the precise role of SHPS-1 and its homologs is not fully understood, it is possible that recruitment of SHP-2 to this membrane protein also results in activation of the Ras-MAP kinase signaling pathway. It is not yet known whether SHP-2 must interact with all three potential targets (the receptor, Gab2, or SHPS-1) to fully execute its positive role in mediating signaling from a single RTK or cytokine receptor. Further studies including identification of substrates will be required to clarify the precise function of SHP-2 and Csw in mitogenic signal trans-duction.

6.3.2. Dual Specificity Phosphatases Negatively Regulate Mitogenic Signaling by Dephosphorylating MAP Kinases

A subset of the DSPs suppresses growth factor and cytokine signaling pathways that are mediated by the MAP kinase cascade. The prototype for this group of DSPs is MKP1 (MAP kinase phosphatase 1) which specifically dephosphorylates both the Thr and Tyr residues located within the activation loop of the MAP kinase catalytic domain. Because both residues must be phosphorylated for MAP kinase to be fully activated, MKP1 has the capacity to inactivate MAP kinase and thereby terminate signaling by this pathway as illustrated in Fig. 10. Expression of MKP1 is induced in response to growth factors and stress (e.g., heat shock, exposure to oxidants, UV irradiation, etc.). The delayed expression of MKP1 following mitogenic stimulation or stress demonstrates that MKP1 acts as a feedback inhibitor of MAP kinase signaling. This function is consistent with the genetics of the Drosophila puckered gene that encodes an MKP1-like enzyme that regulates a MAP kinase homolog in a similar manner.

Besides MKP1, at least nine other related DSPs with activity toward MAP kinases have been identified. More than a dozen MAP kinase signaling modules are present in mammalian cells including the ERK1/2, JNK/SAPK, and p38/RK protein kinases and the existence of multiple MKP1-like phospha-tases may reflect the diversity among these MAP kinases. Several of the MAP kinase phosphatases such as MKP3/PYST1 and PYST2 are expressed constitu-tively and are not induced by stress or growth factors. They are also found in the cytosol instead of the nucleus. Some of the MAP kinase phosphatases are selective and target a subset of the MAP kinases. MKP3/PYST1 exhibits specificity for the ERK kinases, whereas the hVH5/M3-6 phosphatases selectively target the JNK/SAPK kinases that mediate the response of cells to heat, osmotic shock, or UV radiation.

MKP1 and related DSPs are not the exclusive regulators of MAP kinase. In yeast, the tyrosine-specific enzymes, PTP1 and PTP2, are involved in regulating MAP kinases and in some cases both a MAP kinase phosphatase and a PTP may act together to regulate the same kinase. In addition, Ser/Thr protein phosphatases may also act on MAP kinases. These findings suggest that the physiologic regulation of MAP kinases by dephosphorylation is complex and may involve interplay among several types of phosphatases.

6.3.3. Role of Nonreceptor PTPs as Potential Negative Regulators of RTKs

Many other tyrosine-specific PTPs have been implicated in regulating RTKs, cytokine receptors, or the pathways that are downstream of them. For example, biochemical studies have implicated PTP1B, a nontransmembrane PTP, as a negative regulator of insulin receptor signaling. This function has been corroborated by studies of mice in which the PTP1B gene is disrupted. In response to insulin treatment, homozygous PTP1B~'~ mice exhibit increased insulin sensitivity and enhanced phosphorylation of the insulin receptor and its substrate, IRS-1. These knockout mice and biochemical data indicate that the insulin receptor itself may be the PTP1B target, but other targets in the pathway may exist as well. The PTP1B~'~ mice have no other detectable developmental defects and other abnormalities, suggesting that there may be other PTPs that can act as functional replacements or that PTP1B has a narrow role in regulating RTKs.

The T cell PTP (TCPTP), the closest relative of PTP1B, has been implicated in regulating the epidermal growth factor receptor (EGFR). A 45-kDa splice variant of the TCPTP resides in the nucleus, but is translocated to the cytoplasm in response to EGF treatment. Substrate trapping experiments indicate that the EGFR itself and the associated Shc adaptor protein are physiologic substrates. The 45-kDa TCPTP variant inhibited the EGF-induced association of the EGFR with Shc and Grb2, but had no effect on MAP kinase activation. This suggests that this form of TCPTP could act to inhibit or suppress a discrete subset of pathways downstream of the receptor. A distinct 48-kDa variant of the TCPTP resides in the endoplasmic reticulum and may also act on the EGFR, but the physiologic role of this enzyme is not understood.

6.3.4. LMW-PTPs Regulate Receptor Tyrosine Kinases

The LMWPTP is involved in regulating signal transduction through the platelet-derived growth factor (PDGF) RTK. Interestingly, LMWPTP in unstim-ulated NIH3T3 cells is found in the cytoplasm and in association with the cytoskeleton. On PDGF stimu lation, soluble LMWPTP transiently associates with the PDGF receptor. Studies suggest that once bound LMWPTP dephosphorylates the receptor and represses downstream signaling by Src kinase and STAT (signal transducer and activator of transcription) pathways, but not by phospholipase Cy, PI3 kinase, and MAP kinase. In contrast, the cytoskeleton-associated fraction appears to be transiently phos-phorylated and activated by Src kinase, which leads to dephosphorylation of cytoskeletal proteins. LMWPTP in the two subcellular locations responds to PDGF with different kinetics. The cytoskeleton-associated fraction exhibits a maximum response at 40 min, whereas the soluble enzyme responds more rapidly with a maximum response at 5 min. Potential effects of LMWPTP dephosphorylation on the cyto-skeleton are not known. It will be interesting to learn whether there is any overlap between LMWPTP and the classic PTPs in modulating mitogenic signaling.

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