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Evidence for one or more Raf-1 kinase kinase(s) activated by insulin and polypeptide growth factors
R M Lee1, U R Rapp, P J Blackshear
1Howard Hughes Medical Institute Laboratories, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Abstract:
The protein product of the Raf-1 proto-oncogene is a protein serine/threonine kinase that is activated after stimulation of cells with insulin and other mitogens. To investigate the mechanism of this activation, we used purified Raf-1 expressed in E. coli as a substrate for a putative Raf-1 protein kinase kinase. In three different insulin-sensitive cell types, insulin activated Raf-1 kinase kinase activity in crude cytosolic cellular fractions. The insulin stimulation of this activity was evident as early as 2 min after exposure to insulin, maximal at 5-8 min, and inapparent at 15 min. Phosphoamino acid analysis of phosphorylated Raf-1 revealed that serine was the primary phosphate acceptor for the insulin-activated kinase or kinases; small amounts of phosphothreonine were also detected. The insulin effect occurred in cells depleted of protein kinase C, and in extracts depleted of endogenous Raf-1 kinase by immunodepletion; these data argue against protein kinase C or Raf-1 kinase itself being the insulin-stimulated activity. The insulin-activated kinase or kinases phosphorylated the Raf-1 protein on multiple sites in vitro, as evidenced by tryptic mapping; at least some of these appeared to overlap with sites phosphorylated in response to serum in intact cells. Several other mitogens and growth factors stimulated Raf-1 kinase kinase activity, including epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, serum, and phorbol 12-myristate 13-acetate. This insulin- and mitogen-stimulated Raf-1 kinase kinase activity may play a role in mediating the phosphorylation and possibly the activation of the Raf-1 kinase by insulin and other growth factors.
Insights
Insulin and growth factors activate Raf-1 kinase kinase activity, a key step in cell signaling. This enzyme phosphorylates Raf-1 protein, potentially mediating its activation by mitogens.
Area of Science:
- Cellular signaling pathways
- Oncogene research
- Protein kinase cascades
Background:
- Raf-1 proto-oncogene encodes a serine/threonine kinase activated by mitogens.
- Understanding Raf-1 activation mechanisms is crucial for cell growth and cancer research.
Purpose of the Study:
- To investigate the mechanism of Raf-1 kinase activation by insulin.
- To identify the upstream kinase responsible for phosphorylating and activating Raf-1.
Main Methods:
- Used purified bacterial-expressed Raf-1 as a substrate.
- Assayed Raf-1 kinase kinase activity in cytosolic fractions from insulin-stimulated cells.
- Performed phosphoamino acid analysis and tryptic mapping of phosphorylated Raf-1.
Main Results:
- Insulin rapidly activated Raf-1 kinase kinase activity in multiple cell types.
- Serine was the primary phosphorylation site on Raf-1, with some phosphothreonine detected.
- Activity was independent of protein kinase C and endogenous Raf-1 kinase.
Conclusions:
- An insulin-stimulated Raf-1 kinase kinase activity exists and phosphorylates Raf-1.
- This activity may mediate Raf-1 phosphorylation and activation by insulin and other growth factors.
- Identified a potential key regulatory step in mitogenic signaling pathways.