Faculty Bibliography

The protein-tyrosine phosphatase Shp2 plays an essential role in growth factor and integrin signaling, and Shp2 mutations cause developmental defects and/or malignancy. Previous work has placed Shp2 upstream of Ras. However, the mechanism of Shp2 action and its substrate(s) are poorly defined. Additional Shp2 functions downstream of, or parallel to, Ras/Erk activation also are proposed. Here, we show that Shp2 promotes Src family kinase (SFK) activation by regulating the phosphorylation of the Csk regulator PAG/Cbp, thereby controlling Csk access to SFKs. In Shp2-deficient cells, SFK inhibitory C-terminal tyrosines are hyperphosphorylated, and the tyrosyl phosphorylation of multiple SFK substrates, including Plcgamma1, is decreased. Decreased Plcgamma1 phosphorylation leads to defective Ras activation on endomembranes, and may help account for impaired Erk activation in Shp2-deficient cells. Decreased phosphorylation/activation of other SFK substrates may explain additional consequences of Shp2 deficiency, including altered cell spreading, stress fibers, focal adhesions, and motility

Protein tyrosine phosphatase-1b (Ptp1b) inhibits insulin and leptin signaling by dephosphorylating specific tyrosine residues in their activated receptor complexes. Insulin signals are mediated by tyrosine phosphorylation of the insulin receptor and its downstream targets, such as Irs1 and Irs2. Irs2 plays an especially important role in glucose homeostasis because it mediates some peripheral actions of insulin and promotes pancreatic beta-cell function. To determine whether the deletion of Ptp1b compensates for the absence of Irs2, we analyzed mice deficient in both Ptp1b and Irs2. Pancreatic beta-cell area decreased in Ptp1b(-/-) mice, consistent with decreased insulin requirements owing to increased peripheral insulin sensitivity. By contrast, peripheral insulin sensitivity and beta-cell area increased in Irs2(-/-)::Ptp1b(-/-) mice, which improved glucose tolerance in Irs2(-/-)::Ptp1b(-/-) mice and delayed diabetes until 3 months of age. However, beta-cell function eventually failed to compensate for absence of Irs2. Our studies demonstrate a novel role for Ptp1b in regulating beta-cell homeostasis and indicate that Ptp1b deficiency can partially compensate for lack of Irs2.

Nerve growth factor (NGF) mediates the survival and differentiation of neurons by stimulating the tyrosine kinase activity of the TrkA/NGF receptor. Here, we identify SHP-1 as a phosphotyrosine phosphatase that negatively regulates TrkA. SHP-1 formed complexes with TrkA at Y490, and dephosphorylated it at Y674/675. Expression of SHP-1 in sympathetic neurons induced apoptosis and TrkA dephosphorylation. Conversely, inhibition of endogenous SHP-1 with a dominant-inhibitory mutant stimulated basal tyrosine phosphorylation of TrkA, thereby promoting NGF-independent survival and causing sustained and elevated TrkA activation in the presence of NGF. Mice lacking SHP-1 had increased numbers of sympathetic neurons during the period of naturally occurring neuronal cell death, and when cultured, these neurons survived better than wild-type neurons in the absence of NGF. These data indicate that SHP-1 can function as a TrkA phosphatase, controlling both the basal and NGF-regulated level of TrkA activity in neurons, and suggest that SHP-1 regulates neuron number during the developmental cell death period by directly regulating TrkA activity.

The protein-tyrosine phosphatase Shp2 is required for normal activation of the ERK mitogen-activated protein kinase in multiple receptor tyrosine kinase signaling pathways. In fibroblasts, Shp2 undergoes phosphorylation at two C-terminal tyrosyl residues in response to some (fibroblast growth factor and platelet-derived growth factor (PDGF)) but not all (epidermal growth factor and insulin-like growth factor) growth factors. Whereas the catalytic activity of Shp2 is required for all Shp2 actions, the effect of tyrosyl phosphorylation on Shp2 function has been controversial. To clarify the role of Shp2 tyrosyl phosphorylation, we infected Shp2-mutant fibroblasts with retroviruses expressing wild type Shp2 or mutants of either (Y542F or Y580F) or both (Y542F,Y580F) C-terminal tyrosines. Compared with wild type cells, ERK activation was decreased in Y542F- or Y580F-infected cells in response to fibroblast growth factor and PDGF but not the epidermal growth factor. Mutation of both phosphorylation sites resulted in a further decrease in growth factor-evoked ERK activation, although not to the level of the vector control. Immunoblot analyses confirm that Tyr-542 and Tyr-580 are the major sites of Shp2 tyrosyl phosphorylation and that Tyr-542 is the major Grb2 binding site. However, studies with antibodies specific for individual Shp2 phosphorylation sites reveal unexpected complexity in the mechanism of Shp2 tyrosyl phosphorylation by different receptor tyrosine kinases. Moreover, because Y580F mutants retain nearly wild type Grb2-binding ability, yet exhibit defective PDGF-evoked ERK activation, our results show that the association of Grb2 with Shp2 is not sufficient for promoting full ERK activation in response to these growth factors, thereby arguing strongly against the "Grb2-adapter" model of Shp2 action.

Grb2-associated binder 2 (Gab2), a member of the Dos/Gab subfamily scaffolding molecules, plays important roles in regulating the growth, differentiation, and function of many hematopoietic cell types. In this paper, we reveal a novel function of Gab2 in Fcgamma receptor (FcgammaR)-initiated phagocytosis in macrophages. Upon FcgammaR activation, Gab2 becomes tyrosyl phosphorylated and associated with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3K), and the protein-tyrosine phosphatidylinositol Shp-2. FcgammaR-mediated phagocytosis is severely impaired in bone marrow-derived macrophages from Gab2-/- mice. The defect in phagocytosis correlates with decreased FcgammaR-evoked activation of Akt, a downstream target of PI3K. Using confocal fluorescence microscopy, we find that Gab2 is recruited to the nascent phagosome, where de novo PI3K lipid production occurs. Gab2 recruitment requires the pleckstrin homology domain of Gab2 and is sensitive to treatment with the PI3K inhibitor wortmannin. The Grb2 binding site on Gab2 also plays an auxiliary role in recruitment to the phagosome. Because PI3K activity is required for FcgammaR-mediated phagocytosis, our results indicate that Gab2 acts as a key component of FcgammaR-mediated phagocytosis, most likely by amplifying PI3K signaling in the nascent phagosome.

Src homology-2 (SH2) domain-containing phosphatases (Shps) are a small, highly conserved subfamily of protein-tyrosine phosphatases, members of which are present in both vertebrates and invertebrates. The mechanism of regulation of Shps by ligand binding is now well understood. Much is also known about the normal signaling pathways regulated by each Shp and the consequences of Shp deficiency. Recent studies have identified mutations in human Shp2 as the cause of the inherited disorder Noonan syndrome. Shp2 mutations might also contribute to the pathogenesis of some leukemias. In addition, Shp2 might be a key virulence determinant for the important human pathogen Helicobacter pylori. Despite these efforts, however, the key targets of each Shp have remained elusive. Identifying these substrates remains a major challenge for future research.

Tyrosine phosphorylation plays an important role in controlling cellular growth, differentiation and function. Abnormal regulation of tyrosine phosphorylation can result in human diseases such as cancer. A major challenge of signal transduction research is to determine how the initial activation of protein-tyrosine kinases (PTKs) by extracellular stimuli triggers multiple downstream signaling cascades, which ultimately elicit diverse cellular responses. Recent studies reveal that members of the Gab/Dos subfamily of scaffolding adaptor proteins (hereafter, "Gab proteins") play a crucial role in transmitting key signals that control cell growth, differentiation and function from multiple receptors. Here, we review the structure, mechanism of action and function of these interesting molecules in normal biology and disease.

Secretion of leptin from adipocytes communicates body energy status to the brain by activating the leptin receptor long form (LRb). LRb regulates energy homeostasis and neuroendocrine function; the absence of LRb in db/db mice results in obesity, impaired growth, infertility and diabetes. Tyr 1138 of LRb mediates activation of the transcription factor STAT3 during leptin action. To investigate the contribution of STAT3 signalling to leptin action in vivo, we replaced the gene encoding the leptin receptor (lepr) in mice with an allele coding for a replacement of Tyr 1138 in LRb with a serine residue (lepr(S1138)) that specifically disrupts the LRb-STAT3 signal. Here we show that, like db/db mice, lepr(S1138) homozygotes (s/s) are hyperphagic and obese. However, whereas db/db mice are infertile, short and diabetic, s/s mice are fertile, long and less hyperglycaemic. Furthermore, hypothalamic expression of neuropeptide Y (NPY) is elevated in db/db mice but not s/s mice, whereas the hypothalamic melanocortin system is suppressed in both db/db and s/s mice. LRb-STAT3 signalling thus mediates the effects of leptin on melanocortin production and body energy homeostasis, whereas distinct LRb signals regulate NPY and the control of fertility, growth and glucose homeostasis.

Receptor tyrosine kinases (RTKs) are key regulators of cellular homeostasis. Based on in vitro and ex vivo studies, protein tyrosine phosphatase-1B (PTP1B) was implicated in the regulation of several RTKs, yet mice lacking PTP1B show defects mainly in insulin and leptin receptor signaling. To address this apparent paradox, we studied RTK signaling in primary and immortalized fibroblasts from PTP1B(-/-) mice. After growth factor treatment, cells lacking PTP1B exhibit increased and sustained phosphorylation of the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR). However, Erk activation is enhanced only slightly, and there is no increase in Akt activation in PTP1B-deficient cells. Our results show that PTP1B does play a role in regulating EGFR and PDGFR phosphorylation but that other signaling mechanisms can largely compensate for PTP1B deficiency. In-gel phosphatase experiments suggest that other PTPs may help to regulate the EGFR and PDGFR in PTP1B(-/-) fibroblasts. This and other compensatory mechanisms prevent widespread, uncontrolled activation of RTKs in the absence of PTP1B and probably explain the relatively mild effects of PTP1B deletion in mice.