Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 21 (21), 7345-54

Calmodulin Binds to K-Ras, but Not to H- Or N-Ras, and Modulates Its Downstream Signaling

Affiliations

Calmodulin Binds to K-Ras, but Not to H- Or N-Ras, and Modulates Its Downstream Signaling

P Villalonga et al. Mol Cell Biol.

Abstract

Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.

Figures

FIG. 1
FIG. 1
CaM inhibition synergizes with different growth factors to induce ERK1/2 activation. (A) Quiescent Swiss 3T3 cells were incubated overnight with medium containing 0, 0.2, 0.5, or 1% FBS and then treated with the anti-CaM drug W13 (15 μg/ml) or the control drug W12 (15 μg/ml) for 30 min. (B) Quiescent Swiss 3T3 cells were incubated for 30 min with the indicated concentrations of EGF, Bombesin, or PDGF plus W13 (15 μg/ml), W12 (15 μg/ml), or nothing (−). (C) Quiescent Swiss3T3 cells were incubated with PDGF at the indicated concentration and, in the lanes indicated, W7 (25 μM) or trifluoroperazine (TFP) (12.5 and 25 μM) was added. For all panels phosphorylation of ERK1/2 was analyzed by Western blotting using specific anti-P-ERK1/2 antibodies as indicated in Materials and Methods.
FIG. 2
FIG. 2
CaM inhibition induces Ras activation in the absence of any other stimuli. Subconfluent NIH 3T3 cells were incubated for 24 h with 0.5% FBS (A) or serum-free medium (B), and then PDGF (0.4 nM), W13 (15 μg/ml), W12 (15 μg/ml), trifluoroperazine (TFP) (25 μM), or nothing (−) was added to the medium and left for 5 min. The amount of Ras-GTP was analyzed by pull-down assay with RBD-Sepharose and Western blotting using a pan-Ras antibody. Total Ras was also analyzed by Western blotting directly from an aliquot of the corresponding cell lysate. Results from a representative experiment out of five for each condition are shown.
FIG. 3
FIG. 3
CaM inhibition at low FBS concentration does not lead to PKB phosphorylation. Subconfluent NIH 3T3 cells were incubated for 24 h with medium containing 0.5% FBS, and then W13 (15 μg/ml) was added to the medium and left for 5, 10, 20, 30, and 45 min. A negative control (untreated cells) and a positive control (cells treated for 10 min with 10% FBS) were also loaded in the same gel. Total PKB, phosphorylated PKB (P-PKB), and phospho-ERK1/2 were analyzed by Western blotting using specific antibodies.
FIG. 4
FIG. 4
Ca2+-dependent binding of Ras and Raf-1 from cellular lysates to CaM-Sepharose. (A) Cellular lysates (0.5 ml) from NIH 3T3 cells (5 × 106) were incubated with CaM-Sepharose (Seph) in the presence of Ca2+ or EGTA as indicated in Materials and Methods. The presence of Ras, Raf-1, MEK, ERK, GRB-2, p120 GAP, Sos1, and NF1 in the bound and unbound fractions was analyzed by Western blotting using specific antibodies. All bound fraction and 50 μl of the unbound fraction were loaded. (B) As in panel A, but half of the cellular lysate was applied in the presence of Ca2+ to CaM-Sepharose and half was applied to control Sepharose. The presence of Ras and Raf-1 in the bound fractions was analyzed by Western blotting. (C) Cellular lysate (1 ml) was incubated with CaM-Sepharose as indicated in Materials and Methods in the presence of Ca2+. The unbound fraction was collected, and after washing with Ca2+-containing buffer, bound proteins were eluted sequentially (E1, E2, and E3) with 40 μl of the same buffer supplied with EGTA (5 mM). Finally, the remaining bound proteins were eluted with SDS-containing buffer (ESDS). Twenty-five microliters of the unbound fraction and all eluted fraction were loaded onto a SDS-acrylamide gel, and the amount of Ras present in each fraction was analyzed by Western blotting. Pan-Ras antibody was used to detect Ras in all panels.
FIG. 5
FIG. 5
Binding of Ras-GTP from cellular lysates to CaM-Sepharose (Seph) independently of the presence of Raf. (A) Subconfluent NIH 3T3 cells (5 × 106) were incubated for 24 h with medium containing 0.5% FBS and then lysed with 1 ml of lysis buffer. In the indicated lanes, cellular lysates were made with a buffer containing 5 mM MgCl2. CaM pull-down assays were performed with Ca2+ or EGTA, and the presence of Ras and Raf-1 in the bound and unbound fractions was analyzed by Western blotting using specific antibodies. All bound fraction and 25 μl of the unbound fraction were loaded. (B) Subconfluent NIH 3T3 cells were incubated for 24 h with medium containing 0.5% FBS. Cells were lysed with CaM pull-down buffer in the presence or absence of 5 mM MgCl2 and incubated for 1 h at 4°C, and then the amount of Ras-GTP was analyzed by the RBD pull-down method. (C) NIH 3T3 cells were treated with the indicated concentrations of geldanamycin (GA) for 12 h. CaM pull-down assays were performed in the presence of Ca2+ or EGTA. The amounts of Ras and Raf-1 present in the bound and unbound fractions were analyzed by Western blotting using specific antibodies.
FIG. 6
FIG. 6
Ras coimmunoprecipitates with CaM. NIH 3T3 cell extracts were incubated with anti-CaM antibodies (α-CaM) or a nonrelated monoclonal antibody (mAb), and the immunocomplex was pulled down using protein G-Sepharose. The presence of Ras in the immunoprecipitate was analyzed by Western blotting using pan-Ras antibodies. An aliquot of the cellular lysate was also loaded (lane L).
FIG. 7
FIG. 7
Binding of K-RasB but not K-RasA, H-Ras, or N-Ras from cellular lysates to CaM-Sepharose. NIH 3T3 cellular lysates were incubated with CaM-Sepharose (Seph) in the presence of Ca2+ or EGTA. (A) The presence of the different Ras isoforms N-Ras, H-Ras, and K-Ras in the bound and unbound fractions was analyzed by Western blotting using specific antibodies. (B) Same as in panel A, but the Western blot was incubated with either K-RasA or K-RasB antibodies. (C) Fifty-nanogram quantities of H-Ras–GST and K-RasB–GST fusion proteins expressed in Sf9 cells and of bacterially expressed H-Ras, N-Ras, and K-Ras (Oncogene) were loaded onto five different gels, and Western blotting with the indicated antibodies was performed.
FIG. 8
FIG. 8
Preferential activation of K-Ras by CaM inhibition. Subconfluent NIH 3T3 cells were incubated for 24 h with 0.5% FBS, and then PDGF (0.4 nM), W13 (15 μg/ml), W12 (15 μg/ml), or nothing (−) was added to the medium and left for 5 min. The activation of the different Ras isoforms was analyzed by pull-down assay with RBD-Sepharose and Western blotting using antibodies specific for each of the isoforms. (A) Quantification of the scanned Western blots corresponding to three different experiments was performed, and the relationship between the intensities of the bands after W12 or W13 treatment with respect to nontreated cells (Q) is shown. Error bars indicate standard deviations. (B) Results from a representative experiment out of three performed are shown.
FIG. 9
FIG. 9
Binding of purified K-Ras to CaM-Sepharose. (A) Purified GST–H-RasV12 and GST–K-RasBV12 proteins expressed in Sf9 cells were loaded with either GTP or GDP as indicated in Materials and Methods. Proteins were then incubated with CaM-Sepharose (Seph) (in the presence of Ca2+ or EGTA [E]) or with control Sepharose (in the presence of Ca2+). The amounts of proteins in the unbound and bound fractions were analyzed by Western blotting using pan-Ras antibodies. (B) GTP-loaded GST–K-RasBV12 was incubated with CaM-Sepharose in the presence of Ca2+ or EGTA, and in the indicated lane CaM-Sepharose was preincubated with the indicated amounts of CaMKII290–309 peptide in the presence of Ca2+. The amount of Ras in the bound fractions was analyzed by Western blotting using pan-Ras antibodies. (C) CaM-Sepharose or Sepharose control pull-down assays were performed with bacterially expressed and purified GST–K-RasB1–166 and GST–H-Ras1–166 in the presence of Ca2+ or EGTA. The amounts of Ras in the unbound and bound fractions were analyzed by Western blotting using pan-Ras antibodies.

Similar articles

See all similar articles

Cited by 61 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback