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. 1998 Jul;18(7):3947-55.
doi: 10.1128/MCB.18.7.3947.

14-3-3 facilitates Ras-dependent Raf-1 activation in vitro and in vivo

S Roy  1 , R A McPherson, A Apolloni, J Yan, A Lane, J Clyde-Smith, J F Hancock
Affiliations

14-3-3 facilitates Ras-dependent Raf-1 activation in vitro and in vivo

S Roy et al. Mol Cell Biol. 1998 Jul.

Abstract

14-3-3 proteins complex with many signaling molecules, including the Raf-1 kinase. However, the role of 14-3-3 in regulating Raf-1 activity is unclear. We show here that 14-3-3 is bound to Raf-1 in the cytosol but is totally displaced when Raf-1 is recruited to the plasma membrane by oncogenic mutant Ras, in vitro and in vivo. 14-3-3 is also displaced when Raf-1 is targeted to the plasma membrane. When serum-starved cells are stimulated with epidermal growth factor, some recruitment of 14-3-3 to the plasma membrane is evident, but 14-3-3 recruitment correlates with Raf-1 dissociation and inactivation, not with Raf-1 recruitment. In vivo, overexpression of 14-3-3 potentiates the specific activity of membrane-recruited Raf-1 without stably associating with the plasma membrane. In vitro, Raf-1 must be complexed with 14-3-3 for efficient recruitment and activation by oncogenic Ras. Recombinant 14-3-3 facilitates Raf-1 activation by membranes containing oncogenic Ras but reduces the amount of Raf-1 that associates with the membranes. These data demonstrate that the interaction of 14-3-3 with Raf-1 is permissive for recruitment and activation by Ras, that 14-3-3 is displaced upon membrane recruitment, and that 14-3-3 may recycle Raf-1 to the cytosol. A model that rationalizes many of the apparently discrepant observations on the role of 14-3-3 in Raf-1 activation is proposed.

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Figures

FIG. 1
FIG. 1
Plasma membrane-recruited Raf-1 is not complexed with 14-3-3. (A) P100 fractions from COS cells transfected with empty vector (EXV) or an EXV expression plasmid for FLAG RafCAAX or FLAG RafCAAXDD, or cotransfected with EXV expression plasmids for FLAG Raf plus RasG12V or FLAG RafDD plus RasG12V, were normalized for protein content, resolved by SDS-PAGE, and Western blotted for Raf-1 and 14-3-3. (B) S100 and P100 fractions from COS cells transfected with combinations of EXV expression plasmids for FLAG Raf, RasG12V, and myc14-3-3β were normalized for protein content, resolved by SDS-PAGE, and Western blotted for FLAG, Ras, and myc. The Western blots in panels A and B were developed by enhanced chemiluminescence and quantitated by phosphorimaging (Bio-Rad). We conclude that less than 5% of total endogenous 14-3-3, or 2% of myc14-3-3, fractionates with cell membranes, irrespective of the amount of Raf-1 bound to the membranes. (C) S100 and solubilized P100 fractions from COS cells transfected with an EXV expression plasmid for FLAG RafCAAX or cotransfected with EXV expression plasmids for FLAG Raf plus RasG12V were normalized for protein content and immunoprecipitated by using anti-FLAG Sepharose. The anti-FLAG immunoprecipitates (IP) were resolved by SDS-PAGE and Western blotted for Raf and 14-3-3. The amount of EXV RasG12V used in this experiment was 25% of that used in the experiment for which results are shown in panel B, so that only half of the coexpressed FLAG Raf was recruited to the membrane. Only a small amount of RafCAAX can be immunoprecipitated from the cytosol, since most is localized to the plasma membrane.
FIG. 2
FIG. 2
Activated Ras recruits Raf-1 but not 14-3-3 to the plasma membrane. BHK cells were seeded onto coverslips and cotransfected by using Lipofectamine with EXV expression plasmids for myc14-3-3 plus FLAG Raf (A and C) or for myc14-3-3 plus FLAG Raf plus RasG12V (B and D). Duplicate coverslips from each transfection were fixed after 24 h and incubated with anti-FLAG or anti-myc antisera followed by FITC-conjugated anti-mouse immunoglobulin G. The expressed proteins were visualized by indirect immunofluorescence with a confocal microscope. Transfection efficiency was 90%. FLAG Raf and myc14-3-3 both localize to the cytosol in the absence of RasG12V (A and C), but whereas coexpression of RasG12V results in recruitment of FLAG Raf to the plasma membrane (B), 14-3-3 remains in the cytosol (D). Additional control experiments (not shown, but described in Materials and Methods) were performed to visualize transfected Ras and to confirm that the staining patterns of myc14-3-3 and FLAG Raf were no different when they were coexpressed from those obtained when each protein was expressed alone.
FIG. 3
FIG. 3
EGF stimulates transient membrane recruitment of Raf-1 and 14-3-3. COS cells grown for 18 h in serum-free medium were treated for various times (in seconds) with 50 nM EGF. The cells were fractionated, and 10 μg of each P100 fraction was resolved by SDS-PAGE and immunoblotted for Raf-1 and 14-3-3. Immunoblots from a typical representative experiment are shown. There are some Raf-1 and 14-3-3 bound to the P100 fraction at 0 s. To allow pooling of data from independent experiments, the immunoblots were quantitated by phosphorimaging and the fold increases in P100-associated Raf-1 and 14-3-3 were calculated for each time point by using the value at 0 s as 1. Data on seven immunoblots from three independent experiments are presented in the upper graph. Raf-1 activity was assayed on the same membrane fractions in a coupled MEK-ERK assay and is shown in the lower graph.
FIG. 4
FIG. 4
Overexpression of 14-3-3 potentiates the activity of membrane-recruited Raf. (A) Membrane (P100) fractions from COS cells expressing combinations of FLAG Raf, RasG12V, and myc14-3-3 were normalized for FLAG Raf content by quantitative Western blotting (as illustrated in Fig. 1). No FLAG Raf was present in four of the control assays, so a mass of membranes equivalent to that used in the FLAG Raf plus Ras plus 14-3-3 assay was assayed. The Raf-1 kinase activity was measured in a coupled MEK-ERK assay. A representative kinase assay is shown. Each Raf-1 assay was performed both with MEK plus ERK and with ERK alone (to estimate background counts; for details, see Materials and Methods): aliquots of the MBP kinase assay from the MEK-ERK and the control incubation, respectively, are shown alongside each other in the kinase panel. The FLAG immunoblot shown is that of the kinase reaction mixtures containing FLAG Raf; it verifies the Raf-1 assay normalization. (B) Although only a single kinase assay is shown in panel A, each P100 fraction was assayed in triplicate, and each respective MBP kinase assay was performed in duplicate (see Materials and Methods). The mean (n = 6) Raf-1 activities shown have had background counts subtracted. The units are picomoles of phosphate transferred to MBP per 10 min. Results are representative of those obtained in three independent experiments.
FIG. 5
FIG. 5
In vitro activation of Raf-1 by Ras requires 14-3-3. (A) Raf-1 cytosol prepared from COS cells expressing FLAG Raf was mixed on ice with Ras membranes prepared from COS cells expressing RasG12V or with control membranes from COS cells transfected with empty EXV plasmid. To control for endogenous Raf-1 already associated with the EXV and Ras membranes, incubations were also set up with buffer A in place of the Raf-1 cytosol. Either 6 μg of recombinant 14-3-3 (1 mg/ml in buffer A), 6 μl of peptide pS-Raf 621 (500 μM in buffer A), or 6 μl of buffer A was added, the samples were agitated for 10 min at 25°C, and membranes were reisolated by centrifugation. The immunoblots show the amounts of Raf-1 (detected by anti-FLAG) and 14-3-3 bound to the P100 fractions after the 10-min incubation. The Raf-1 activity associated with the membranes was measured in a coupled MEK-ERK assay as described in Materials and Methods. (B) FLAG Raf was immunoprecipitated with anti-FLAG Sepharose from Raf-1 cytosol containing no peptide (lane 1), 40 μM Raf-621 (nonphosphorylated Raf peptide) (lane 2), or 40 μM pS-Raf-621 (phosphorylated Raf peptide) (lane 3) and was immunoblotted for Raf-1 and 14-3-3. The 40 μM concentration was selected after a set of preliminary titration experiments using peptide concentrations in the range of 1 to 100 μM (33a). IP, immunoprecipitate. (C) The Raf-1 blots and the coupled MEK-ERK assay for membrane-associated Raf-1 activity in panel A were quantitated by phosphorimaging. Ras-dependent Raf-1 binding was calculated from the phosphorimager data by subtracting the value obtained for Raf-1 bound to control EXV membranes (treated as background). For the calculation of Raf-1 specific activities, the Raf-1 activity associated with EXV or Ras membranes after incubation in buffer A was subtracted from the Raf-1 activity associated with the respective membranes after incubation with Raf-1 cytosol to obtain the net membrane-dependent Raf-1 activation. The Raf-1 specific activity was then calculated by dividing the net Raf-1 activity by the amount of Raf-1 recruited. The total amount of 14-3-3 associated with the P100 fractions was also estimated by phosphorimaging. The amount of 14-3-3 bound to membranes was 50% greater for the Raf-plus-Ras incubation, and 30% lower for the incubation with peptide, than the amount present in Ras membranes incubated with buffer A. There was no overall increase in the amount of membrane-bound 14-3-3 when recombinant 14-3-3 was included in the incubation, although endogenous 14-3-3 was partially replaced by recombinant 14-3-3 (which has a slightly slower mobility). Similar changes were also seen in the amount of 14-3-3 bound to the control membranes.
FIG. 6
FIG. 6
Raf-1 activated by Ras in vitro is not complexed with 14-3-3. Raf-1 cytosol prepared from COS cells expressing FLAG Raf was mixed on ice with Ras membranes prepared from COS cells expressing RasG12V or with control membranes from COS cells transfected with empty EXV plasmid. EXV and Ras membranes were also incubated with buffer A in place of the Raf-1 cytosol. Given that only small amounts of Raf-1 associate with the EXV membranes (see Fig. 5), fivefold more control membranes and Raf-1 cytosol were used in the EXV incubations than in the Ras membrane incubations. Either 6 μg of recombinant 14-3-3 (1 mg/ml in buffer A), 6 μl of peptide pS-Raf 621 (500 μM in buffer A), or 6 μl of buffer A was added, the samples were agitated for 10 min at 25°C, and membranes were reisolated by centrifugation and solubilized by sonication in 1% NP-40. The FLAG Raf bound to the membranes was immunopurified with anti-FLAG Sepharose and assayed for kinase activity in a coupled MEK-ERK assay. After the kinase assay, the FLAG immunoprecipitates were immunoblotted for Raf-1 (by using anti-Raf-1 antisera) and 14-3-3. Note that Raf-1 recruited to the membranes by Ras is activated and is devoid of 14-3-3, whereas Raf-1 that has bound nonspecifically to the EXV membranes is not activated and remains complexed with 14-3-3. The Raf-1 activity associated with Ras membranes in this assay is much lower than that seen in Fig. 5 because endogenous Raf-1 is not captured by the anti-FLAG Sepharose. Quantification of the Raf-1 immunoblots and the Raf-1 kinase assay by phosphorimaging showed that the amount of FLAG Raf-1 associated with the Ras membranes decreased by 60% in the presence of peptide pS-621 and by 40% in the presence of recombinant 14-3-3. There was a fivefold stimulation of Raf-1 specific activity by recombinant 14-3-3 and a 60% inhibition of Raf-1 specific activity by peptide pS-Raf621. These figures are comparable with those derived in Fig. 5C.
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