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Molecular and Cellular Biology, April 2002, p. 2375-2387, Vol. 22, No. 7
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.7.2375-2387.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Protein Phosphatase 2A Forms a Molecular Complex with Shc and Regulates Shc Tyrosine Phosphorylation and Downstream Mitogenic Signaling

Satoshi Ugi, Takeshi Imamura, William Ricketts,,{dagger} and Jerrold M. Olefsky*

Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0673; San Diego Veterans Affairs Hospital, Research Service, San Diego, California 92161; and The Whittier Diabetes Institute, La Jolla, California 92037

Received 30 August 2001/ Returned for modification 2 October 2001/ Accepted 18 December 2001

Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase that carries out multiple functions. Although numerous observations suggest that PP2A plays a major role in downregulation of the mitogen-activated protein (MAP) kinase pathway, the precise mechanisms are unknown. To clarify the role of PP2A in growth factor (insulin, epidermal growth factor [EGF], and insulin-like growth factor 1 [IGF-1]) stimulation of the Ras/MAP kinase pathway, simian virus 40 small t antigen was expressed in Rat-1 fibroblasts which overexpress insulin receptors. Small t antigen is known to specifically inhibit PP2A by binding to the A PP2A regulatory subunit, interfering with the ability of PP2A to bind to its cellular substrates. Overexpressed small t protein was coimmunoprecipitated with PP2A and inhibited cellular PP2A activity but did not inhibit protein phosphatase 1 (PP1) activity. Insulin, IGF-1, and EGF stimulation also inhibited PP2A activity. Growth factor-stimulated Ras, Raf-1, MAP kinase, and mitogen-activated extracellular-signal-regulated kinase kinase (MEK) activities were elevated in small-t-antigen-expressing cells. Furthermore, Shc tyrosine phosphorylation and its association with Grb2 were also elevated in small-t-antigen-expressing cells. Expression levels of Shc, Ras, MEK, or MAP kinase and phosphorylation of insulin, EGF, and IGF-1 receptors were not altered. Interestingly, we found that PP2A associated with Shc in the basal state and dissociated in response to insulin and EGF and that this dissociation was inhibited by 65% in small-t-antigen-expressing cells. In addition, we found that PP2A associates with the phosphotyrosine-binding domain (PTB domain) of Shc and that phosphorylation of tyrosine 317 of Shc was required for PP2A-Shc dissociation. We conclude (i) that PP2A negatively regulates the Ras/MAP kinase pathway by binding to Shc, inhibiting tyrosine phosphorylation; (ii) that the Shc-PP2A association is mediated by the Shc PTB domain but the interaction is independent of phosphotyrosine binding, indicating a new molecular function for the PTB domain; (iii) that growth factor stimulation, or small-t-antigen expression, causes dissociation of the PP2A-Shc complex, facilitating Shc phosphorylation and downstream activations of the Ras/MAP kinase pathway; and (iv) that this defines a new mechanism of small-t-antigen action to promote mitogenesis.

* Corresponding author. Mailing address: Department of Medicine (0673), University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0673. Phone: (858) 534-6651. Fax: (858) 534-6653. E-mail: jolefsky{at}ucsd.edu.

{dagger} Present address: Isis Pharmaceuticals, Inc., Carlsbad, CA 92008.

Molecular and Cellular Biology, April 2002, p. 2375-2387, Vol. 22, No. 7
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.7.2375-2387.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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