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Tyrosine-phosphorylated SOCS-3 inhibits STAT activation but binds to p120 RasGAP and activates Ras

N A Cacalano1, D Sanden, J A Johnston

  • 1DNAX Research Institute, 901 California Avenue, Palo Alto, California 94304, USA.

Nature Cell Biology
|May 2, 2001
PubMed

Insights

Suppressors of cytokine signalling (SOCS) proteins regulate cell responses. Tyrosine phosphorylation of SOCS-3 surprisingly promotes cell survival and proliferation via the Ras pathway, despite inhibiting other growth factor signals.

Area of Science:

  • Cellular signaling
  • Molecular biology
  • Immunology

Background:

  • Suppressors of cytokine signalling (SOCS) proteins, including SOCS-3, are immediate early genes involved in negative feedback regulation of cytokine signaling pathways.
  • SOCS proteins inhibit the activation of signal transducer and activator of transcription (STAT) factors, thereby dampening cellular responses to cytokines.
  • Understanding the precise mechanisms by which SOCS proteins modulate signaling pathways is crucial for comprehending cellular growth and survival regulation.

Purpose of the Study:

  • To investigate the role of tyrosine phosphorylation of SOCS-3 in response to various growth factors.
  • To elucidate the interaction of phosphorylated SOCS-3 with downstream signaling molecules, specifically p120 RasGAP.
  • To determine the impact of SOCS-3 phosphorylation status on the activation of STAT and extracellular-signal-regulated kinase (ERK) pathways and subsequent cell proliferation.

Main Methods:

  • Western blotting to detect tyrosine phosphorylation of SOCS-3 in response to interleukin-2 (IL-2), erythropoietin (EPO), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF).
  • Co-immunoprecipitation assays to assess the interaction between phosphorylated SOCS-3 and p120 RasGAP.
  • Analysis of STAT5 and ERK activation using phospho-specific antibodies after cytokine stimulation in cells expressing wild-type or tyrosine mutant SOCS-3.
  • Cell proliferation assays to evaluate the effect of SOCS-3 phosphorylation on IL-2, EPO, and PDGF-induced proliferation.

Main Results:

  • SOCS-3 undergoes strong tyrosine phosphorylation upon stimulation with IL-2, EPO, EGF, and PDGF.
  • Phosphorylation at residues 204 and 221 of SOCS-3 facilitates interaction with p120 RasGAP.
  • Phosphorylated SOCS-3 inhibits STAT5 activation but maintains ERK activation via RasGAP binding following IL-2 stimulation.
  • A tyrosine mutant of SOCS-3 blocked STAT phosphorylation and also strongly inhibited IL-2-, EPO-, and PDGF-induced ERK activation and cell proliferation.

Conclusions:

  • Tyrosine phosphorylation of SOCS-3 plays a dual role: inhibiting STAT signaling while promoting Ras/ERK pathway activation.
  • The interaction of phosphorylated SOCS-3 with RasGAP is critical for maintaining ERK activation and promoting cell proliferation.
  • Despite the general inhibitory role of SOCS proteins, tyrosine phosphorylation of SOCS-3 paradoxically supports cell survival and proliferation through the Ras pathway.

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