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. 2014 May 2;289(18):12886-95.
doi: 10.1074/jbc.M114.561613. Epub 2014 Mar 13.

Complex formation between S100B protein and the p90 ribosomal S6 kinase (RSK) in malignant melanoma is calcium-dependent and inhibits extracellular signal-regulated kinase (ERK)-mediated phosphorylation of RSK

Kira G Hartman  1 , Michele I Vitolo, Adam D Pierce, Jennifer M Fox, Paul Shapiro, Stuart S Martin, Paul T Wilder, David J Weber
Affiliations

Complex formation between S100B protein and the p90 ribosomal S6 kinase (RSK) in malignant melanoma is calcium-dependent and inhibits extracellular signal-regulated kinase (ERK)-mediated phosphorylation of RSK

Kira G Hartman et al. J Biol Chem. .

Abstract

S100B is a prognostic marker for malignant melanoma. Increasing S100B levels are predictive of advancing disease stage, increased recurrence, and low overall survival in malignant melanoma patients. Using S100B overexpression and shRNA(S100B) knockdown studies in melanoma cell lines, elevated S100B was found to enhance cell viability and modulate MAPK signaling by binding directly to the p90 ribosomal S6 kinase (RSK). S100B-RSK complex formation was shown to be Ca(2+)-dependent and to block ERK-dependent phosphorylation of RSK, at Thr-573, in its C-terminal kinase domain. Additionally, the overexpression of S100B sequesters RSK into the cytosol and prevents it from acting on nuclear targets. Thus, elevated S100B contributes to abnormal ERK/RSK signaling and increased cell survival in malignant melanoma.

Keywords: Calcium; Calcium Binding Proteins; Cancer; ERK; MAP Kinases (MAPKs); Melanoma; RSK; S100 Proteins.

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Figures

FIGURE 1.
FIGURE 1.
S100B increases melanoma cell viability. A, Western blot analysis of total cell lysates (25 μg) showing the level of S100B expression of the non-targeting scrambled WM115 cells (lane S) as well as two S100B knockdown clonal lines (inset, lanes 1 and 2). Cellular viability of the knockdown cells compared with that of the non-targeting scrambled cells was assessed at several time points over the course of 7 days using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay to determine the change in cell number. The scrambled control cells (S), S100B knockdown clone 1 (1), and clone 2 (2) are also represented as in the inset. Shown is a compilation of three different experiments, each performed in triplicate (n = 9). B, Western blot analysis was performed on total cell lysate (25 μg) to confirm expression of S100B in the 501mel cells. Shown are 501mel cells containing vector alone (V) and the S100B clonal lines A and B (inset). Cellular viability of the 501mel S100B-expressing clonal lines was compared with that of the vector control cells at several time points measured over a period of 7 days. The vector control cells (V), S100B-expressing clone A (A), and clone B (B) are represented as in the inset. Shown is a compilation of three different experiments, each performed in triplicate (n = 9).
FIGURE 2.
FIGURE 2.
S100B suppresses RSK Thr-573 phosphorylation by ERK. A, Western blot analysis (25 μg) of WM115 cells with the non-targeting scrambled vector (S) and two stable S100B knockdown clonal cell lines derived from WM115 cells (1 and 2). B, Western blot analysis (20 μg) showing the effects of S100B expression in 501mel cells (first and second lanes) compared with an empty vector control (third and fourth lanes) in the absence (first and third lanes) or presence (second and fourth lanes) of the MEK1/2 inhibitor (5 μg/ml) U0126.
FIGURE 3.
FIGURE 3.
Inhibition of RSK phosphorylation by S100B is Ca2+-dependent. A, in vitro kinase assays examining the ERK-mediated phosphorylation of RSK Thr-573 in the absence and presence of S100B and 1 mm CaCl2. Western blot analysis was employed following a 15-min incubation period, and changes in pRSK at Thr-573 were observed. B, Western blot analysis of 25 μg of 501mel cell lysate with untreated cells (first lane) or cells transfected with empty vector (second lane), a vector with wild-type S100B (third lane), or a vector with the E31A/E72A double mutant of S100B (fourth lane), which is an S100B construct incapable of binding calcium (18). These data confirm that S100B must bind calcium ions to bind and inhibit RSK phosphorylation at Thr-573.
FIGURE 4.
FIGURE 4.
S100B directly binds to RSK and a construct of RSK with the CTKD but not to ERK. A, schematic of RSK1 (residues 1–735) showing the N-terminal kinase domain (NTKD, residues 62–321), the CTKD (residues 418–675), the ERK binding site at the C terminus of RSK1 (residues 722–735, underlined), and the phosphorylation sites reported to be necessary for RSK activation are labeled P (43). B, Western blot analysis of RSK and ERK protein levels from BSA or S100B pulldown eluates performed in the presence of either 5 mm EDTA (first through third lanes) or 5 mm CaCl2 (fourth through sixth lanes). The first and fourth lanes contain total WM115 cell lysate antibody controls (CT), whereas eluates of BSA and lysate are shown in the second and fifth lanes, and S100B and lysate are shown in the third and sixth lanes. C, Western blot analysis of GST or GST-RSK1386–752 cell-free pulldowns supplemented with either 5 mm EDTA (second through fifth lanes) or 5 mm calcium (sixth through ninth lanes). Eluates of GST with S100B (second and sixth lanes), GST-RSK1386–752 with S100B (third and seventh lanes), GST with the E31A/E72A mutant (fourth and eighth lanes), and GST-RSK1386–752 with the E31A/E72A mutant (fifth and ninth lanes) are shown. S100B protein was loaded in the first lane as an antibody control.
FIGURE 5.
FIGURE 5.
RSK nuclear localization is inhibited by S100B. A, Western blot analysis comparing the cytoplasmic (C) and nuclear (N) protein levels of S100B knockdown clone 1 (1) to those of the non-targeting scrambled cell line (S). Shown is an example experiment that was repeated in triplicate. The nuclear matrix protein p84 and MEK were used as loading controls for the nuclear and cytoplasmic fractions, respectively. B, immunofluorescence studies showing the localization of pRSK Thr-573 in both the S100B knockdown clone 1 and non-targeting scrambled cell lines (representative of three different fields of vision are shown). C, immunofluorescence studies focusing on a single XYZ image to show the localization of pRSK Thr-573 as well as nucleolar exclusion for each cell line. Planes are indicated by dotted lines and examples of nucleolar exclusion by arrows (representative of numerous XYZ images).
FIGURE 6.
FIGURE 6.
Schematic representation of the Ca2+-dependent effects of S100B on the MAPK signaling cascade. S100B-Ca2+ directly interacts with RSK, inhibiting phosphorylation at Thr-573 by ERK and reducing subsequent translocation of RSK to the nucleus, thus allowing it to act on its cytoplasmic targets but not on nuclear targets. Phosphorylation sites on RSK are labeled with the letter P.
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