Protein tyrosine phosphatase α (PTPα) is believed to dephosphorylate physiologically the Src protooncogene at phosphotyrosine (pTyr)527, a critical negative-regulatory residue. It thereby activates Src, and PTPα overexpression neoplastically transforms NIH 3T3 cells. pTyr789 in PTPα is constitutively phosphorylated and binds Grb2, an interaction that may inhibit PTPα activity. We show here that this phosphorylation also specifically enables PTPα to dephosphorylate pTyr527. Tyr789→Phe mutation abrogates PTPα-Src binding, dephosphorylation of pTyr527 (although not of other substrates), and neoplastic transformation by overexpressed PTPα in vivo. We suggest that pTyr789 enables pTyr527 dephosphorylation by a pilot binding with the Src SH2 domain that displaces the intramolecular pTyr527-SH2 binding. Consistent with model predictions, we find that excess SH2 domains can disrupt PTPα-Src binding and can block PTPα-mediated dephosphorylation and activation in proportion to their affinity for pTyr789. Moreover, we show that, as predicted by the model, catalytically defective PTPα has reduced Src binding in vivo. The displacement mechanism provides another potential control point for physiological regulation of Src-family signal transduction pathways.

A high rate of aerobic glycolysis was catalyzed by rat-i cells transfected with a ras oncogene (ras cells); rat-i cells and rat-i cells transtected with myc oncogene (myc cells) showed a low rate of glycolysis that was increased after exposure of the cells to type B transforming growth factor (TGF-P). The uptake of radioactive methylaminoisobutyric acid or L-methionine via system A of amino acid transport also was accelerated after exposure of these cells to TGF-p, with the myc cells being most sensitive and the ras cells least sensitive. Methionine was found to be a potent inhibitor of glycolysis in ras cells as well as In rat-i or myc cells that were exposed to TGF-P. We propose a relationship between the product of the ras oncogene (p21) and the protein(s) induced by exposure to TGF-,B.In a landmark paper (1) We have observed (9) that normal rat kidney NRK-49F cells exhibited on exposure to type 3 transforming growth factor (TGF-,B) an increase in the rate of glycolysis and in the uptake of methylaminoisobutyrate (MeAIB), a specific substrate of system A of amino acid transport (10). Independently, Inman and Colowick showed that glucose uptake was stimulated by TGF-/3 in 3T3 cells (11). We also have reported (9) that glycolysis in NRK-49F cells that had been exposed to TGF-P was markedly inhibited by 25 mM methionine, a substrate for system A of amino acid transport, whereas little or no inhibition was noted in the controls that were not exposed to TGF-f3. Examination of various established transformed cell lines revealed that in all of them glycolysis was sensitive to methionine, whereas several nontransformed cell lines showed no or moderate inhibition (unpublished data). Although there were differences between cell lines with respect to the time of exposure to methionine (2-16 hr) required to give rise to an inhibition of 50% or more, thus far no exception has been observed among 10 transformed cell lines grown in tissue culture that were tested. However, glycolysis in suspended EAT cells harvested from infected mice was not inhibited by methionine under the conditions tested. Yet, EAT cells grown in tissue culture were among the most sensitive cell lines, showing a significant inhibition after only 30 min exposure to 10 mM methionine. Removal of methionine from the medium resulted in a complete reversal of the inhibition within 2 hr (unpublished data). The capacity to inhibit glycolysis was shared by other substrates transported via system A, including MeAIB, a synthetic compound that is not metabolized.In view of the fact that methionine inhibits glycolysis in transformed cell lines, we explored the effect of this amino acid and of TGF-,B on rat-1 cells and rat-1 variants transfected with ras or myc oncogenes. We describe in this paper some profound differences in glycolysis, MeAIB uptake, and response to TGF-/3 that we observed between these cell lines. MATERIALS AND METHODSThe rat-i, myc (R1-CMYC), and ras (R1-EJ2) cells were obtained from R. Weinberg. They were grown in Falcon dishes Abbrevia...

We show that siRNA-mediated suppression of protein tyrosine phosphatase a (PTPa) reduces Src activity 2 to 4-fold in breast, colon and other human cancer cell lines. Src and PTPa RNAi induced apoptosis in estrogen receptor (ER)-negative breast cancer and colon cancer cells, but not in immortalized noncancerous breast cells, ER-positive breast cancer cells or other cancer cell types tested. RNAi of other Src family members (Fyn and Yes) or of PTP1B, a phosphatase previously suggested to be an activator of Src in breast cancer, had no effect. Although further tests with primary tumor tissues are required, the unexpected correlation between ER status and Src/PTPa dependence in breast cancer cell lines may be important for planning therapeutic strategies, and the insensitivity of normal breast cells to the RNAi highlights the potential of PTPa, which may be easier to target than Src, as a therapeutic target in ER-negative breast cancer.

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During mitosis, the catalytic activity of protein-tyrosine phosphatase (PTP) ␣ is enhanced, and its inhibitory binding to Grb2, which specifically blocks Src dephosphorylation, is decreased. These effects act synergistically to activate Src in mitosis. We show here that these effects are abrogated by mutation of Ser 180 Protein-tyrosine phosphatase (PTP)1 ␣ is an ϳ130-kDa transmembrane PTP (1, 2) that activates the cytoplasmic membrane-bound Src protein-tyrosine kinase by dephosphorylating Src Tyr(P) 527 (Refs. 3 and 4; see Ref. 5 for review). This releases Src from its negatively regulated conformation in which Tyr (P) 527 is bound intramolecularly to the Src SH2 domain (see Refs. 6 and 7 for review). Overexpression of PTP␣ results in dephosphorylation of Tyr(P) 527 and activation of Src in vivo (3, 4). Conversely, Src Tyr(P) 527 phosphorylation is higher and Src catalytic activity is about three times lower in cells from PTP␣ Ϫ/Ϫ knockout mice (8, 9) or following antisense-induced PTP␣ down-regulation (10), indicating that PTP␣ is a major physiological positive regulator of Src. Constitutive activation of the Src proto-oncoprotein by mutation increases the tyrosine phosphorylation of multiple signal transduction proteins and thereby neoplastically transforms a variety of cell types (see Ref. 7 for review). The fact that activation by overexpressed PTP␣ also causes transformation (3) is perhaps more surprising and suggests that PTP␣ activity is directed in vivo preferentially to Src (and Src family members), rather than to Src substrates.This substrate specificity is due, at least in part, to a phosphotyrosine displacement mechanism that selectively promotes dephosphorylation of Src by PTP␣: ϳ20% of PTP␣ in NIH3T3 cells is phosphorylated at Tyr 789 , a residue near its carboxyl terminus (11, 12). Tyr 789 phosphorylation does not affect PTP␣ dephosphorylation of nonspecific substrates such as myelin basic protein (MBP), whose phosphotyrosines are not bound, but is required for dephosphorylation of Src Tyr(P) 527 , which is protected against many phosphatases by its SH2 domain binding (13). Phosphorylated Tyr 789 can bind to the Src SH2 domain, thereby displacing and thus unprotecting Src Tyr(P) 527 . This also forms a transient bound state that additionally facilitates Tyr(P) 527 dephosphorylation (13). Tyr (P) 789 also binds the SH2 domain of the adapter protein Grb2 (11, 12), which participates in Ras activation following peptide growth factor stimulation (see Ref. 14 for review). Because of steric hindrance resulting from the interaction of one of the Grb2 SH3 domains with PTP␣, PTP␣-bound Grb2 is not able to bind Sos, the downstream protein in the Grb2-Ras signal transduction pathway. Thus, it does not appear that localization of Grb2 to the plasma membrane by binding to PTP␣ can activate the Ras signaling pathway (15, 16). Instead, control may flow in the other direction: Grb2 binding to Tyr(P) 789 blocks phosphotyrosine displacement and the ability of PTP␣ to dephosphorylate Src, so only Grb2-unbo...