Autoregulation of the Raf-1 serine/threonine kinase

Abstract

The Raf-1 serine/threonine kinase is a key protein involved in the transmission of many growth and developmental signals. In this report, we show that autoinhibition mediated by the noncatalytic, N-terminal regulatory region of Raf-1 is an important mechanism regulating Raf-1 function. The inhibition of the regulatory region occurs, at least in part, through binding interactions involving the cysteine-rich domain. Events that disrupt this autoinhibition, such as mutation of the cysteine-rich domain or a mutation mimicking an activating phosphorylation event (Y340D), alleviate the repression of the regulatory region and increase Raf-1 activity. Based on the striking similarites between the autoregulation of the serine/threonine kinases protein kinase C, Byr2, and Raf-1, we propose that relief of autorepression and activation at the plasma membrane is an evolutionarily conserved mechanism of kinase regulation.

Figure 1

Reg/Raf inhibits the biological and enzymatic activity of Cat/Raf. (A) Schematic depiction of the Reg/Raf and Cat/Raf proteins. (B) Effect of Reg/Raf on Xenopus oocyte meiotic maturation. Oocytes were injected with buffer (−) or RNA encoding the Reg/Raf protein (+). Four to eight hours later, the oocytes were injected with RNA encoding Cat/Raf-1, Ha-Ras^V12, Tpr-Met, or v-Mos or were treated with progesterone (Prog). GVBD was then scored. Numbers shown represent a compilation of at least five (Cat/Raf) or two (Ha-Ras^V12, Tpr-Met, v-Mos, Prog) independent experiments where equivalent amounts of Reg/Raf proteins were expressed. (C) Reg/Raf inhibits the enzymatic activity of Cat/Raf and the activation of MEK1 and MAPK. Cat/Raf (Top), MEK1 (Middle), and MAPK (Bottom) proteins were immunoprecipitated from Xenopus oocytes expressing Cat/Raf alone (−) or coexpressing Cat/Raf and Reg/Raf (+), and in vitro protein kinase assays were performed. The positions of MEK1, glutathione S-transferase (GST)-MAPK, or myelin basic protein (MBP), used as exogenous substrates in these assays, are indicated.

Figure 2

Association of the Reg/Raf and Cat/Raf proteins. FLAG immunoprecipitates were prepared from Xenopus oocytes expressing Cat/Raf alone (−) or coexpressing Cat/Raf and FLAG-tagged Reg/Raf (+). The immunoprecipitates were resolved by electrophoresis on an SDS/8% polyacrylamide gel and examined for the presence of Cat/Raf by immunoblotting with antibodies directed against the C-terminus of Raf-1 (Top). Total oocyte lysates were analyzed by immunoblotting to evaluate the expression level of FLAG-tagged Reg/Raf (Middle), and Cat/Raf (Bottom).

Figure 3

Regulatory region mutations alter the inhibitory effect of Reg/Raf. (A) Effects of regulatory region mutations on the activity of full-length Raf-1 proteins. Oocytes were injected with RNA (30 ng) encoding full-length WT-, R89L-, CRM-, F163I-, or P181L-Raf-1 and scored for GVBD. (B) Effects of regulatory region mutations on Reg/Raf inhibition of Cat/Raf. Oocytes were first injected with buffer (Control) or RNA encoding WT-, R89L-, CRM-, F163I-, or P181L-Reg/Raf. Four to eight hours later, oocytes were injected with RNA encoding Cat/Raf and then scored for GVBD. Oocyte lysates were analyzed by immunoblotting to evaluate the expression level of FLAG-tagged Reg/Raf (Middle) and Cat/Raf-1 (Bottom). (C) Effects of regulatory region mutations on the inhibition of Ha-Ras^V12 by Reg/Raf. Oocytes were injected with buffer (Control) or RNA encoding WT-, R89L-, CRM-, F163I-, or P181L-Reg/Raf. Four to eight hours later, oocytes were injected with RNA encoding Ha-Ras^V12 and then scored for GVBD. Oocyte lysates were analyzed by immunoblotting to evaluate the expression level of FLAG-tagged Reg/Raf (Middle) and Ha-Ras^V12 (Bottom). The numbers shown in A, B, and C represent a compilation of three independent experiments; the protein analysis shown in B and C is from one typical experiment.

Figure 4

Binding of 14–3-3 to WT and mutant Reg/Raf proteins. FLAG-tagged Reg/Raf proteins were immunoprecipitated from oocytes lysed in RIPA buffer. The immunoprecipitates were washed extensively and then incubated for 2 hr with extracts from 293 cells (as a source of exogenous 14–3-3). The immunoprecipitates were examined by immunoblot analysis by using FLAG (Upper) and 14–3-3 (Lower) antibodies.

Figure 5

Activating mutations in Cat/Raf prevent Reg/Raf suppression. Oocytes were injected with buffer (−) or RNA encoding Reg/Raf (+). Four to eight hours later, oocytes were injected with RNA encoding WT-, YY340,341FF-, or Y340D-Cat/Raf and then scored for GVBD. Oocyte lysates were analyzed by immunoblotting to evaluate the expression level of FLAG-tagged Reg/Raf (Middle) and Cat/Raf (Bottom). Numbers shown represent a compilation of three independent experiments where equivalent amounts of Reg/Raf and Cat/Raf proteins were expressed; the protein analysis is from one typical experiment.

Figure 6

Model of Raf-1 regulation. (A) In quiescent cells, Ras is in the inactive GDP-bound form and Raf-1 is in an inactive state in the cytosol. The regulatory region inhibits the activity of the catalytic domain, perhaps through intramolecular interactions involving the CRD (Upper). Under signaling conditions, Ras becomes GTP-loaded and activated. Raf-1 translocates to the plasma membrane, where both the RBD and CRD contact membrane components, thereby relieving the repression by the regulatory region (Lower). (B) In the absence of Ras activation, activating mutations in the CRD (Upper) or the catalytic domain (Lower) relieve the repression of the regulatory region and allow the catalytic domain to contact its downstream target, MEK1.