Cell Type-Specific Regulation of B-Raf Kinase by cAMP and 14-3-3 Proteins

and MAPK of B-Raf by cAMP with enzyme-associated 14-3-3 and the cAMP-mediated inhibition prevented by over-expression of 14-3-3; expression of a dominant negative 14-3-3 resulted in partial loss of B-Raf kinase activity. Our data suggest a model in which 14-3-3 protects B-Raf from A-kinase inhibition and may explain B-Raf’s varying response to and 2) or an expression vector encoding 14-3-3$ (lanes as described in Methods. as was probed first with an antibody specific for of with of corresponding to Ser728 in the catalytic domain of B-Raf [upper designated (19,49,53). The which does not distinguish phospho-and dephospho-forms, and Similar results were obtained in two other experiments.


INTRODUCTION
The serine/threonine protein kinases of the Raf family (Raf-1, A-Raf and B-Raf) are key regulators of cell growth, differentiation and apoptosis in eukaryotic cells (1). They are activated by a large number of membrane receptors which stimulate Raf kinases indirectly, though small GTP-binding proteins of the Ras family (1)(2)(3). Activated Raf kinases phosphorylate and activate the dual-specificity kinases MEK-1 and -2 which in turn phosphorylate and activate the mitogen-activated protein kinases (MAPKs) 1 Erk-1 and -2.
Raf-1 is expressed ubiquitously, but A-Raf and B-Raf are differentially expressed with highest levels in urogenital tissues and brain, respectively (1). The Raf kinases differ in their response to upstream signals and their ability to activate the MAPK pathway (4)(5)(6)(7)(8).
Raf-1 activation requires phosphorylation on Ser 338 and Tyr 340 subsequent to Ras•GTP binding; binding of Rap1•GTP to Raf-1 does not lead to activation (6,(9)(10)(11)(12). In B-Raf, the serine residue equivalent to Ser 338 of Raf-1 is constitutively phosphorylated and the residue equivalent to Tyr 340 of Raf-1 is an aspartate, leading to high basal activity of B-Raf compared to Raf-1; B-Raf can be activated fully by binding to Ras•GTP or Rap•GTP (4,6,(12)(13)(14). Although both B-Raf and Raf-1 are expressed and activated by growth factors in neuronal cells, B-Raf seems to be the major MEK activator in these cells and possibly also in other cell types (4,7,(15)(16)(17)(18). Two major isoforms of B-Raf of 68 and 95 kDa differ by 115 amino acids at the N-terminus; alternative splicing of exons 8 and 10 yields additional isoforms which differ in their tissue distribution, basal MEK kinase activity and oncogenic properties (15,19).
However, a recent report suggests that A-kinase inhibits growth factor-induced Raf-1 activation in 293 human embryonal kidney cells independently of direct Raf-1 phosphorylation (32).
However, several reports showed that cAMP inhibits B-Raf activity in PC12 cells expressing both 95 and 68 kDa B-Raf with one report showing that cAMP inhibits B-Raf in serum-starved cells but not in cells maintained in serum-containing medium (18,34,35,46). Inhibition of B-Raf by cAMP was also described in unstimulated and phorbol ester-stimulated NIH3T3 cells and in chemoattractant-stimulated neutrophils; cAMP-mediated activation of a 68 kDa B-Raf isoform was observed in CHO cells and correlated with Rap1 activation (5,39,47). These results suggest that B-Raf regulation by cAMP may differ depending on growth conditions, expression of different B-Raf isoforms and presence of cell type-specific factors.
A-kinase phosphorylates B-Raf in vitro and in vivo, although the phosphorylation sites have not been mapped (46,48). B-Raf has no equivalent of Raf-1 Ser 43 , but Ser 728 in B-Raf (numbers correspond to the 95 kDa isoform) corresponds to Ser 621 of Raf-1 and the surrounding sequences are highly homologous suggesting that B-Raf Ser 728 may also be a target for A-kinase phosphorylation (19,49). In co-transfection experiments, A-kinase activated full-length B-Raf but inhibited the isolated catalytic domain expressed in PC12 cells; when incubated with B-Raf in vitro, A-kinase had no effect on the activity of the full length enzyme but reduced the activity of the catalytic domain, suggesting that the N-terminal regulatory domain of B-Raf prevents A-kinase from inhibiting B-Raf catalytic activity (48).
The family of 14-3-3 proteins includes at least seven isoforms which are abundantly expressed in most tissues and bind as homo-or heterodimers to phosphoserine residues in the consensus sequence RSXpSXP (50)(51)(52). Raf-1 contains at least three 14-3-3 binding sites: one in the cysteine-rich domain between amino acids 136 and 187, a second surrounding Ser 259 , and a third surrounding Ser 621 (50,(53)(54)(55). Mutations in the two N-terminal sites which prevent 14-3-3 binding lead to Raf-1 activation (54,55); Ras•GTP and phosphatidylserine binding near these sites displaces bound 14-3-3 allowing full activation of Raf-1 via phosphorylation of Ser 338 and Tyr 340 and reassociation of 14-3-3 may be involved in returning Raf-1 to the inactive form (52,(56)(57)(58). In contrast, binding of 14-3-3 to Ser 621 of Raf-1 appears to be required for basal kinase activity, because: (i) mutation of Ser 621 to any other residue destroys catalytic activity; (ii) removal of 14-3-3 from the catalytic domain of Raf-1 using specific detergents or competitive phospho-peptides completely abrogates kinase activity which is restored upon adding 14-3-3; and (iii) expression of a dominant negative 14-3-3 results in inhibition of the Raf-1 catalytic domain (53). These data have led to a model in which 14-3-3 binding to Raf-1 is necessary to keep the enzyme in an inactive, but activationcompetent conformation (56,59,60). Some investigators have reported that over-expression of 14-3-3 potentiates Raf-1 activation while others have found no effect (53,54,56,60). All three 14-3-3 binding sites of Raf-1 are highly conserved in B-Raf and intracellularly, B-Raf appears to exists in a high molecular weight complex with 14-3-3 proteins, HSP90 and MEK-1 and -2 (8,49,50,(61)(62)(63). Ser 728 of B-Raf is a 14-3-3 binding site which appears to be necessary for and NB2A cells. We found that inhibition of B-Raf by cAMP correlated with significantly lower amounts of enzyme-associated 14-3-3 and the cAMP-mediated inhibition was completely prevented by over-expression of 14-3-3; expression of a dominant negative 14-3-3 resulted in partial loss of B-Raf kinase activity. Our data suggest a model in which 14-3-3 protects B-Raf from A-kinase inhibition and may explain B-Raf's varying response to cAMP in different cell types.

Assessment of MAPK activation
Cells were cultured for 48 h in either full (10%) serum-containing media or in low serumcontaining media prior to adding 250 µM 8-CPT-cAMP for the indicated time. Cells were lysed in sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis (PAGE) sample buffer and Western blots were generated as described previously (68) using an anti-active MAPK antibody which specifically recognizes the dually phosphorylated, active form of Erk-1 and -2 (69).
Equal loading of protein was verified by reprobing the blot with an Erk-1 and -2-specific antibody. Western blots were developed using horse radish peroxidase-coupled secondary antibodies and enhanced chemiluminescence. In some experiments, MAPK activity was measured in Erk-1/2 immunoprecipitates using myelin basic protein as substrate (68).

Measurement of Rap1-bound GTP and Total Rap1-bound Guanine Nucleotides
Rap1•GTP was captured using RBD peptide (65) and GTP eluted from the isolated Rap was measured as described previously (70); total Rap1-bound guanine nucleotides were measured after converting Rap•GDP to Rap•GTP in half of the sample. This method, which has been described recently (71), correlated well with the method of Franke et al. (65) in which Rap1"GTP bound to RBD peptide is assessed by immunoblotting using a Rap1-specific antibody. Briefly, -3 x 10 6 cells were extracted in a HEPES-based buffer containing 0.92 % Triton X-114 and 0.08 % Triton X-45; to one half of the sample was added 20 mM MgSO 4 and to the other half was added 10 µM GTP and 10 mM EDTA to fully exchange GDP for GTP (72).
After shaking 10 min at 4° C, extracts were warmed to 15° C for 1 min and centrifuged at room temperature at 10,000 x g for 2 min to generate an aqueous and detergent phase. The detergent phase, containing >95% of the Rap1, was diluted 10-fold with lysis buffer lacking detergent but containing 20 mM MgSO 4 and the phase separation was repeated one more time. The diluted detergent phase was then added to glutathione Sepharose beads pre-loaded with 80 µg of GSTtagged RBD peptide and the mixture was shaken gently for 1 h at 4° C to allow quantitative binding of Rap1•GTP to the RBD peptide (65,71). The beads were washed four times and then heated for 3 min at 100° C to elute the GTP bound to Rap1 which was measured using a coupled enzymatic assay (70). The affinity of the RBD peptide for Rap•GDP is undetectably low and we showed that the Ras•GTP present in cell lysates does not interfere with the Rap1 assay, probably because the affinity of RBD for Rap1•GTP is 100-fold higher than for Ras•GTP (71,73). In control experiments using purified Rap1, we found that the in vitro GTP exchange reaction was complete under the described conditions (71). The activation state of Rap1 was calculated as the amount of GTP bound to Rap1 determined in the sample which did not undergo exchange, divided by the total amount of nucleotide-bound Rap1determined in the sample subjected to the exchange reaction.

DNA Determination
DNA was measured in the nuclear fraction by a standard fluorescence method using the fluorescent dye bisbenzimidazole (74)

Immunoprecipitation and B-Raf Kinase Assay
Cells were cultured in full serum-containing media or in low serum-containing media for 48 h before harvesting and were lysed in a HEPES-based buffer containing 1% Triton X-100, protease and phosphatase inhibitors. B-Raf was either immunoprecipitated using a B-Rafspecific antibody and Protein A agarose beads as described previously (68), or for cells transfected with GST-tagged B-Raf, the protein was isolated by incubation with glutathione Sepharose beads. After washing the beads, they were incubated with 300 ng of recombinant MEK-1 and 125 µM [(-32 PO 4 ]ATP for 5 min at 30°C as described previously (68); control experiments indicated the assay was linear with respect to lysate input and time. Reaction products were subjected to SDS-PAGE, electroblotted onto polyvinylidene fluoride membranes and exposed to X-ray film. The amount of B-Raf present in the precipitates was determined by immunoblotting with a B-Raf-specific antibody. Control reactions were performed using precipitates obtained either with Protein A and control rabbit immunoglobulin or with glutathione Sepharose and mock-transfected cells.

Determination of 14-3-3 Association with B-Raf
Cells were transfected with pcDNA3-BRaf or pCMV-GST-BRaf and after 48 h of culture in full serum-containing media, cells were lysed and B-Raf was immunoprecipitated or isolated on glutathione Sepharose beads as described above. Beads were washed four times in lysis buffer and eluted in SDS-PAGE sample buffer. Proteins eluted from the beads and 5% of the input cell lysates were analyzed by SDS-PAGE/Western blotting. For untagged B-Raf immunoprecipitates, blots were first developed using an antibody which recognizes all major 14-3-3 isoforms and then re-probed with a B-Raf-specific antibody. For GST-tagged B-Raf, blots were simultaneously developed with 14-3-3-and B-Raf-specific antibodies.   (33,34,39,41). Assays in which Erk-1 and -2 were immunoprecipitated from cells and MAPK activity was determined using myelin basic protein as substrate confirmed the results shown in Fig. 1 (data not shown).

Cell Type-Specific Regulation of the MAPK Pathway by 8-pCPT-cAMP
The effect of 8-pCPT-cAMP was maximal at 250 µM and 1 mM 8-Br-cAMP or 20 µM forskolin yielded similar results (data not shown).

Effect of 8-pCPT-cAMP on Rap1 Activation
MAPK activation by cAMP in PC12 cells is mediated by Rap1, but Rap1 activation by cAMP appears to be cell type-specific (75,76). Since differential activation of Rap1 by cAMP could explain differences in MAPK regulation by cAMP, we measured the effect of 8-CPT-cAMP on Rap1 activation in the four cell types under study using a novel, quantitative enzymatic method (71).

B-Raf Expression
Multiple, alternatively spliced B-Raf isoforms have been described which may vary in their ability to activate MEK and, therefore, MAPK (15,19,49,78). We compared endogenous B-Raf expression in the different cell types by Western blotting (Fig. 3, upper panel). Although the amounts of the 95 and 68 kDa B-Raf isoforms differed to some extent between the different cell lines, the 95 kDa isoform was present in all four cell lines with NB2A and PC12 cells expressing similar levels. Duplicate blots were probed with an antibody to actin (Fig. 3, middle panel) and with an antibody which recognizes all major isoforms of 14-3-3 (Fig. 3, lower panel); these blots demonstrated equal protein loading and comparable levels of 14-3-3 in all lanes, respectively. Treating cells for 15 min with 8-pCPT-cAMP did not affect B-Raf or 14-3-3 levels (Fig. 3). Apparently, not all C6 cells express B-Raf (27), and in different strains of CHO and PC12 cells varying amounts of the 68 kDa or 95 kDa B-Raf isoform are present (33,35,39,79).

Cell Type-Specific Regulation of B-Raf by 8-pCPT-cAMP
We  (Fig. 6 B). We chose cell lysates which contained comparable amounts of GSTtagged B-Raf for the pull-down assay, and found significantly more 14-3-3 associated with B-Raf in CHO-K1 and PC12 cells compared to C6 and NB2A cells (Fig. 6 B). The differences in the amounts of 14-3-3 associated with B-Raf were not due to differences in B-Raf expression in successfully transfected cells, because transfection efficiencies were similar for C6 and PC12 cells and for CHO-K1 and NB2A cells, respectively (data not shown). Results of three independent experiments, in which the signal intensities for B-Raf and 14-3-3 were quantitated by laser densitometry, indicate that about 5-fold more 14-3-3 protein was associated with B-

Raf in cells in which cAMP stimulated B-Raf activity compared to cells in which cAMP
inhibited B-Raf activity, although the amount of total 14-3-3 protein expressed was similar in all cell types studied (Fig. 6 C and Fig. 3). The amount of 14-3-3 associated with B-Raf appears to be regulated by cell type-specific factors. Similar amounts of total14-3-3 were present in the four cell lines we studied, but significantly less 14-3-3 was associated with B-Raf in cells in which cAMP inhibited B-Raf compared to cells in which cAMP simulated B-Raf. Only a small amount of total cellular 14-3-3 was associated with B-Raf even in PC-12 or CHO-K1 cells in which B-Raf was overexpressed. Cell type-specific differences in the subcellular localization of B-Raf and 14-3-3 could potentially limit association of the two proteins (6,9,51). Numerous other 14-3-3 target proteins, including the protein kinase BCR and the death agonist BAD, may compete with B-Raf for 14-3-3 binding (52,85). Recently, the product of the early response gene BRF1 has been demonstrated to interact tightly with 14-3-3$ and J and to interfere with the binding of 14-3-3

Effect of 14-3-3 on the Regulation of B-Raf Activity by 8-pCPT-cAMP
to Raf-1 (89). Thus, B-Raf association with 14-3-3 proteins may vary between different cell types due to the expression of proteins which enhance or diminish 14-3-3 binding to B-Raf or due to expression of different 14-3-3 isoforms which vary in their affinities for specific target proteins (51,89). More work is clearly necessary to examine the factors which influence 14-3-   with an antibody which recognizes all major isoforms of 14-3-3 (lower panel). Note that at least two different 14-3-3 isoforms were resolved on this 10% acrylamide gel.