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Phosphorylation of Raf-1 by p21-activated kinase 1 and Src regulates Raf-1 autoinhibition

Nancy H Tran1, Jeffrey A Frost

  • 1Department of Integrative Biology and Pharmacology, University of Texas Houston Health Science Center, Houston, Texas 77030, USA.

The Journal of Biological Chemistry
|January 29, 2003
PubMed

Insights

The Raf-1 autoinhibitory domain is functional in mammalian cells, blocking kinase activity. Phosphorylation at specific sites (Serine 338, Tyrosines 340/341) relieves this inhibition, regulating Raf-1 signaling.

Area of Science:

  • Cellular signaling pathways
  • Protein kinase regulation
  • Molecular biology

Background:

  • Mitogen and growth factor stimulation activates Raf-1 kinase through complex mechanisms.
  • The Raf-1 N-terminus contains an autoinhibitory domain, previously shown to inhibit activity in Xenopus oocytes.

Purpose of the Study:

  • To investigate the function of the Raf-1 autoinhibitory domain in mammalian cells.
  • To determine the role of specific phosphorylation events in regulating Raf-1 autoinhibition.

Main Methods:

  • Expression of the Raf-1 autoinhibitory domain in mammalian cells.
  • Assessing extracellular signal-regulated kinase 2 (ERK2) activation.
  • Investigating the effects of PAK1 and Src phosphorylation on Raf-1 activity and domain interactions.

Main Results:

  • The Raf-1 autoinhibitory domain blocked ERK2 activation by the Raf-1 catalytic domain in mammalian cells.
  • Phosphorylation of Raf-1 on Serine 338 (by PAK1) and Tyrosines 340/341 (by Src) relieved autoinhibition.
  • This relief of autoinhibition correlated with decreased binding between the Raf-1 regulatory and catalytic domains.
  • Phosphorylation of Threonine 491 and Serine 494 did not appear to regulate autoinhibition.

Conclusions:

  • The Raf-1 autoinhibitory domain is functional in mammalian cells.
  • Phosphorylation at Serine 338 and Tyrosines 340/341 regulates the interaction between the Raf-1 autoinhibitory and catalytic domains, thereby controlling Raf-1 activity.

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