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Laboratory of Cancer Pharmacogenetics, Van Andel Research Institute, Grand Rapids, Michigan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The family of 90-kDa serine/threonine RSKs (p90RSK or MAPK-activated protein kinase 1) was among the first MAPK substrates identified (11) . In humans, four separate genes encoding four isoforms (RSK1, 2, 3, and 4) were identified (11, 12, 13) . Mutations in RSK2 are associated with Coffin-Lowry syndrome, an X-linked disorder characterized by mental retardation and skeletal abnormalities (12) . RSKs have two distinct kinase domains and are activated by ERK and 3-phosphoinositide-dependent protein kinase 1 binding to and subsequent phosphorylation of RSK, as well as by autophosphorylation driven by the RSK COOH-terminal kinase domain (14, 15, 16, 17, 18, 19) . RSKs phosphorylate a variety of substrates via the NH2-terminal kinase domain, including the transcriptional factors cAMP-responsive element-binding protein, c-Fos, and Myt1 (20, 21, 22) , and they regulate a diverse array of cellular functions such as gene transcription, protein synthesis, and cell cycle regulation (23) . More recently, RSK was demonstrated to promote cell survival through phosphorylation and inactivation of the proapoptotic Bcl-2 family member Bad (22 , 24) . However, a role for RSK as an important downstream MAPK effector promoting survival in cancer cells has not been demonstrated.
The Bcl-2 family proteins are key regulators of apoptosis. Three subfamilies have been identified: (a) the prosurvival Bcl-2 (e.g., Bcl-2 and Bcl-XL) proteins; (b) the proapoptotic Bax (e.g., Bax and Bak) proteins; and (c) the BH3 domain-only (e.g., Bad, Bim, and Bid) proteins (25, 26, 27) . Structurally, the BH3-only proteins are divergent from other Bcl-2 family members because they contain only one of the four conserved BH motifs (BH1–4). Normal cellular homeostasis requires the suppression of proapoptotic players by various mechanisms, including phosphorylation, intracellular localization, and heterodimerization with prosurvival Bcl-2 family proteins. Disruption of the balance between pro- and antiapoptotic Bcl-2 family members is suggested to be fundamental to the development of many diseases.
The BH3-only proapoptotic protein Bad is regulated through its phosphorylation and cytosolic sequestration. Dephosphorylated Bad promotes apoptosis by binding to either Bcl-2 or Bcl-XL and titrating them away from proapoptotic Bax/Bak proteins. Unbound Bax oligomerizes and then disrupts mitochondrial integrity, causing cytochrome c release and initiating the caspase cascade (26 , 28) . However, phosphorylated Bad is bound and sequestered in the cytosol by the chaperone protein 14-3-3. Phosphorylation of one of at least three serine residues inactivates Bad by regulating interactions with either 14-3-3 or Bcl-2 family members: Ser75 (human designation; murine equivalent, Ser112); Ser99 (Ser136); or Ser118 (Ser155). Several survival kinases are implicated in the direct phosphorylation of Ser75, Ser99, and Ser118, including RSKs, Akt or p70S6K and PKA, respectively, indicating that Bad functions as an important convergence point in signal transduction pathways affecting cell survival. While RSK-mediated Ser75 phosphorylation or Akt- and/or p70S6K-driven Ser99 phosphorylation creates a consensus motif important for 14-3-3 binding and cytosolic sequestration (29, 30, 31, 32) , PKA-mediated Ser118 phosphorylation is proposed to induce conformational changes in the BH3 domain that disrupt interactions between Bad and prosurvival Bcl-2 family proteins (33, 34, 35) . It is currently unclear which phosphorylation event(s) or which kinase(s) is important in regulating Bad activity in tumor cells.
In this study, we examined the importance of the downstream MAPK pathway effector proteins RSK and Bad in melanoma cell survival. Unlike in normal melanocytes, the MEK/ERK/RSK signaling module was constitutively hyperactivated and was required for melanoma cell survival. RSK-mediated Bad inactivation through Ser75 phosphorylation was necessary for maintaining cell survival in melanoma cells but not in melanocytes, in which an alternative MAPK-independent mechanism(s) may be responsible for survival. Our results identify MEK/ERK/RSK-mediated Bad inactivation as a critical mechanism promoting cell survival specifically in melanoma cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies to phospho-Bad Ser75 (7E11), phospho-RSK1/3 (T359/S363), caspase-9, PARP, phospho-ERK1/2, phospho-MEK1/2, and total MEK1/2 were obtained from Cell Signaling Technology. Antibodies to phospho-Bad Ser118, total Bad (H-168), total ERK1/2, and RSK3 (C20) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to RSK1 and RSK2 were obtained from Upstate Biotechnology. Antibody to phospho-Bad Ser99 was obtained from Biosource (Camarillo, CA). Antibody suitable for RSK1 immunoprecipitation was obtained from R&D Technologies (Minneapolis, MN). 
-Tubulin antibody (DM1A) was obtained from Sigma.
Immunoprecipitation and Western Blotting.
Lysates were prepared in SSB buffer (0.5% NP40, 0.1% Brij-35, 0.1% sodium deoxycholate, 1 mM EDTA, 7 mM K2HPO4, 3 mM KH2PO4, 5 mM EDTA, 10 mM MgCl2, 50 mM ß-glycerol phosphate, 1 mM NaVO4, 2 mM DTT, 5 µg/ml pepstatin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Total Bad antibodies conjugated to protein A beads (36)
were used for immunoprecipitation. Total input protein for each immunoprecipitation reaction was 300 µg, and approximately 1.5 µg of Bad antibody was used per reaction. After overnight immunoprecipitation, beads were washed three times in SSB buffer and boiled in SDS sample buffer for subsequent Western blot analysis. Western blotting was performed as described previously (9)
.
Apoptosis Assays.
Apoptosis was quantitated by either 4',6-diamidino-2-phenylindole staining (9)
or Annexin-V Alexa Fluor-568 (Molecular Probes, Inc., Eugene, OR) binding following the manufacturer’s recommended protocol. Flow cytometry was performed in a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA).
RSK1 Cloning and RT-PCR Analysis.
Wt human RSK1 cDNA was cloned from normal melanocytes by RT-PCR into the TOPO Zero Blunt vector (Invitrogen Corp., Carlsbad, CA), and the sequence was verified with GenBank NM_002953.2. PCR-based site-directed mutagenesis was used to generate RSK1 mutants (Fig. 4A)
. CAI mutant contains an NH2-terminal myristoylation signal (MGSSKSKPK) and the Y702A substitution (24)
, as well as phospho-mimic substitutions EED359 and EDD363 in the regulatory linker region (17
, 37)
. CAII lacks the first 43 NH2-terminal residues but contains Y702A and the phospho-mimic substitutions as described in CAI (17
, 37
, 38)
. A KD mutant harbors S221A and T573A substitutions in catalytic domains and K94A and K447A substitutions in ATP-binding domains (16
, 17
, 24
, 37
, 38)
. All RSK1 and control LacZ constructs were subcloned into pShuttle-cytomegalovirus IRES-GFP (Stratagene, La Jolla, CA) for transfection experiments.
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Transfections and CD4 Selection.
Transfections were performed using LipofectAMINE 2000 Reagent (Invitrogen Corp.) following the manufacturer’s recommended protocol, with minor modifications. Cells were transfected overnight and washed in cold PBS, and growth media were added. For CD4 cotransfections, a 2:1 ratio DNA dilution of the experimental vector to the pMACS 4-IRES.II CD4-encoding vector (Miltenyi Biotec, Auburn, CA) was used. Melanoma cells cotransfected with CD4-expressing vectors were purified using magnetic beads coated with anti-CD4 antibodies using the manufacturer’s recommended protocol (Dynal Biotech, Lake Success, NY). For inhibitor treatments, selected cells were plated overnight in growth media and treated with PD98059 as described above.
Retroviral Vectors and VSV-G Pseudotyped Virus Production.
An IRES-GFP fragment was subcloned into the Moloney murine leukemia virus-based retroviral vector pLPCX (Becton Dickinson, Franklin Lakes, NJ). All RSK1 and control LacZ constructs were subcloned into the resulting vector such that GFP expression is dependent on RSK or LacZ expression. Each vector was transfected into the helper line 293-GPG (a kind gift from Dr. Cindy Miranti; Van Andel Research Institute) to produce VSV-G pseudotyped replication-defective viral stocks. Viral titers were determined on HEK293 cells to ensure equal multiplicity of infection when infecting melanoma cell lines.
RSK1 in Vitro Kinase Assays.
RSK in vitro kinase assays were performed as described previously (24)
with minor modifications. RSK1 immunoprecipitations were performed overnight with rotation at 4°C using 300 µg of total input protein. Kinase assays were performed using 0.25 µg of GST-mouse Bad fusion protein as a substrate.
Stable Bad Transfectants.
Wt human Bad or alanine-substituted Bad (S75A) cDNA was cloned into pDEST26 vector through GATEWAY cloning technology (Invitrogen Corp.). M14-MEL melanoma cells were transfected with human Bad Wt, human Bad S75A, or empty pDEST26 vector using LipofectAMINE 2000 Reagent (Invitrogen Corp.), and pools of stable transfectants were generated by selecting for G418 resistance.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. As described previously (9)
, although comparable levels of MEK1/2 and ERK1/2 expression were detected in melanoma cells and melanocytes, activation of both MEK1/2 and ERK1/2 was relatively higher in melanoma cells. Furthermore, consistent with previous findings (10)
, ERK1/2 activation was constitutive in all melanoma cell lines tested because activated ERK1/2 were still detected in melanoma cells cultured in low (0.1%) serum (Fig. 1C)
. We next evaluated the expression and activation of RSKs, which were previously implicated in MAPK-mediated cell survival (18
, 21
, 22
, 24
, 38
, 40
, 41)
. Preliminary RT-PCR analysis using isoform-specific primers followed by Western blotting showed that three isoforms (RSK1, 2, and 3) were detected in the melanocytes and melanoma cells examined;2
however, among the RSKs analyzed, RSK1 was overexpressed in some and hyperactivated in all melanoma cell lines tested (Fig. 1B)
. Thus, these results demonstrate that the MEK1/2-ERK1/2-RSK1 signaling module is constitutively hyperactivated in melanoma cell lines relative to cultured normal melanocytes. There was no additional ERK1/2 or RSK1 activation induced with the higher (5%) serum content in melanocytes (Fig. 1D)
, indicating that the differential MEK1/2-ERK1/2-RSK1 activation in melanoma cells versus melanocytes was not attributable to the differences in serum content in their respective culture media (Fig. 1, C and D)
.
The proapoptotic function of the Bcl-2 family protein Bad is regulated by its phosphorylation state and is a crucial effector of many survival kinases, including RSKs (22
, 24
, 42, 43, 44)
. We next determined the phosphorylation state of Bad in melanocytes and melanoma cell lines. Bad was highly phosphorylated at Ser75 in both melanoma cells and melanocytes (Fig. 1E)
. However, whereas Bad was phosphorylated at Ser99 and Ser118 in melanocytes, these phosphorylations were minimally detected in melanoma cell lines, with the exception of UACC-62 (Fig. 1E)
. Similar levels of Bad protein expression were found in melanocytes and melanoma cells (Fig. 1E)
.
Bad Phosphorylation at Ser75 Is Dependent on ERK/RSK Activation in Melanoma Cells but not in Normal Melanocytes.
To determine whether RSK activation and Bad phosphorylation are dependent on MAPK signaling, we treated normal melanocytes and melanoma cell lines with MEK inhibitors, either PD98059 or PD184352. In melanoma cells, ERK1/2 and RSK activation as well as Bad Ser75 phosphorylation were inhibited after treatment with either PD98059 or PD184352 (Fig. 2
and data not shown). Bad Ser99 and Ser118 phosphorylations were minimally detected and not sensitive to MEK inhibition in all melanoma cells except UACC-62 cells (Fig. 2, B and C)
. In sharp contrast, ERK1/2-RSK activation (although minimal) and Bad phosphorylation (Ser75, Ser99, and Ser118) were largely unaffected by MEK inhibitors in normal melanocytes (Fig. 2)
, suggesting MAPK-independent regulation of Bad phosphorylation. Collectively, these data suggest a strong MAPK-dependent component to melanoma survival signaling through Bad Ser75 phosphorylation. However, in UACC-62 cells, Bad was phosphorylated at all three serine residues (Figs. 1C
and 2B)
, and Ser99 phosphorylation was also sensitive, in part, to MEK inhibition (Fig. 2B)
, suggesting cross-talk between the MAPK pathway and alternative/additional survival pathways. In fact, significantly enhanced activation of additional survival kinases including Akt, p70S6K, and 3-phosphoinositide-dependent protein kinase 1 was detected in UACC-62 cells when compared with other melanoma cell lines examined (data not shown).
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. PD98059 treatment had slightly delayed and less dramatic apoptotic effects on MALME-3M cells (Fig. 3C)
. Within 4 h of treatment, both ERK1/2 and RSK activation were inhibited in M14-MEL cells (Fig. 3B)
. Bad Ser75 phosphorylation was diminished within 8 h of treatment and was not detected after 36 h. The gradual loss of Bad Ser75 phosphorylation correlated with the onset of caspase-mediated death signaling (25, 26, 27)
in M14-MEL cells, as evidenced by activation of caspase-9 and subsequent cleavage of a caspase-3 substrate, PARP (Fig. 3B)
. Similarly, RSK activation in MALME-3M melanoma cells was inhibited within 4 h of treatment; the loss of Bad Ser75 phosphorylation was evident only after 36–48 h of treatment and was associated with increased apoptosis (Fig. 3, C and D)
. These results suggest that loss of Bad Ser75 phosphorylation (i.e., activation) is consistent with the initiation of MEK inhibition-induced apoptotic response in melanoma cells.
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on apoptosis induced by MEK inhibition. A LacZ expression construct served as a negative control in these experiments. Expression and catalytic activities of RSK1 constructs were confirmed by transient expression in HEK293 cells (Fig. 4B)
and by in vitro kinase assays using recombinant GST-mouse Bad fusion protein as a substrate. Although overexpressing LacZ did not enhance Bad Ser112 (Ser75 equivalent in human Bad) phosphorylation beyond background (GST-Bad alone), overexpressing Wt RSK1 induced significant Bad Ser112 phosphorylation. Expressing CAI or CAII RSK1 yielded further enhanced Bad Ser112 phosphorylation when compared with Wt, although KD RSK1 was unable to phosphorylate Bad Ser112 beyond LacZ levels. Comparable RSK1 expression was observed in all transfectants (Fig. 4B
, bottom panel). We then confirmed the activities of RSK1 constructs toward endogenous Bad in M14-MEL cells. Because of extremely low transfection efficiencies observed in melanoma cells (data not shown), we cotransfected RSK1 constructs with a vector expressing a CD4 cell surface antigen, and the resulting transfectants were enriched using magnetic beads coated with anti-CD4 antibody. The enriched transfectants were then treated with or without PD98059, and the phosphorylation of Bad Ser75 was evaluated. In this transient transfection system, CAI and CAII RSK1-expressing melanoma cells sustained Bad Ser75 phosphorylation in the presence of PD98059, whereas in LacZ-expressing cells, Bad Ser75 phosphorylation was blocked (Fig. 4C)
. Although overexpressed Wt RSK1 induced Bad Ser112 phosphorylation in the in vitro kinase assay (Fig. 4B)
, Bad Ser75 phosphorylation was not detected in M14-MEL cells expressing Wt RSK1 and treated with PD98059 (Fig. 4C)
. This discrepancy may be attributed to experimental difficulties such as low transfection efficiencies and uneven loading. Overexpression of KD RSK1 did not affect Bad Ser75 phosphorylation in untreated cells (Fig. 4C)
.
To evaluate the effects of expressing RSK1 constructs on melanoma cell survival, we used a VSV-G envelope protein pseudotyped Moloney murine leukemia virus-based retroviral vector system with an IRES-GFP expression cassette (45)
. This system allowed for enhanced gene transfer efficiency through infection, and infected cells were identified by GFP coexpression. RSK1 expression was confirmed in M14-MEL cells, with each infection yielding comparable levels of RSK1 expression (data not shown). Infected M14-MEL and MALME-3M cells were treated with PD98059 and analyzed by flow cytometry for apoptosis. Overexpression of Wt, CAI, or CAII RSK1 partially protected both M14-MEL and MALME-3M cells from apoptosis induced by PD98059, whereas KD RSK1 was ineffective (Fig. 4, D and E)
. Similar levels of apoptosis were induced after PD98059 treatment in both LacZ-infected cells and mock-infected cells (Fig. 4E)
. Because the infection efficiencies were still fairly low, the GFP-positive population was enriched after infection by selecting for puromycin resistance (carried in the retroviral vector). RSK1 expression was clearly detected in these enriched populations (Fig. 4F)
. PD98059 treatment induced significant apoptosis in mock- and LacZ-infected cells (Fig. 4F)
. Wt RSK1 expression reduced PD98059-induced cell death by nearly 50%, whereas either CAI or CAII RSK1 expression reduced apoptosis by >75% (Fig. 4F)
. Overexpressing KD RSK1 did not affect overall PD98059-induced apoptosis (Fig. 4F)
. These results, therefore, implicate RSK in mediating a melanoma-specific survival signal.
Bad(S75A) Mutant Further Sensitizes Melanoma Cells to MEK Inhibitor-Induced Apoptosis.
If Bad Ser75 phosphorylation functions in melanoma cell survival, alanine substitution of Ser75 would constitutively activate Bad, enhancing the apoptotic response to MEK inhibition. To evaluate whether Bad(S75A) mutant expression sensitizes melanoma cells to apoptosis induced by a MEK inhibitor, we established stable pools of M14-MEL cells expressing either Wt human Bad or Bad(S75A). We found severalfold greater expression of Wt Bad than Bad(S75A) in these stable lines (Fig. 5A)
. Expression of Bad(S75A) significantly sensitized M14-MEL cells to PD98059-induced apoptosis compared with control-transfected cells (Fig. 5B)
. Sensitization to PD98059 was dose dependent because cells expressing Bad(S75A) were approximately twice as sensitive as control-transfected cells to apoptosis induced by 20 µM PD98059, whereas only moderate sensitization was observed at 5 µM PD98059 (Fig. 5B)
. Although a trend was noted, sensitization by Wt Bad was not statistically significant because of comparatively large SDs (Fig. 5B)
. Moreover, Wt Bad expression was severalfold greater than that of Bad(S75A) (Fig. 5A)
; hence, with protein levels normalized to apoptosis, Bad(S75A) was significantly more potent than Wt Bad in enhancing apoptotic sensitivity of melanoma cells to PD98059 (Fig. 5B)
. Taken together, these results indicate that MAPK/RSK-dependent Bad inactivation by Ser75 phosphorylation is an important mediator of melanoma survival signaling. However, the finding that Bad(S75A) expression is tolerated by melanoma cells (Fig. 5A)
suggests that inactivation of Bad alone may not be sufficient for melanoma cell survival. It is conceivable that additional survival signaling molecules are required to fully confer melanoma cell survival. Furthermore, we cannot rule out the possibility that these stable transfectants may have independently developed an adaptive mechanism to overcome the selective pressure of Bad-induced apoptosis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Constitutive activation of MAPK signaling is a hallmark of many human cancers, including breast and colon cancers and melanoma (1 , 3 , 39 , 46) . Constitutive activation of both MEK and MAPK was demonstrated in melanoma but not in benign nevi (2 , 8) . Expression of CA MEK in immortalized melanocytes promotes tumor formation in nude mice (8) . Recently, a high incidence of activating mutations in B-Raf, an upstream activator of the MEK/ERK signaling, has been found in melanoma cell lines, atypical nevi, and primary tumors (4 , 6 , 10 , 47) , indicating the potential importance of B-Raf and downstream MAPK signaling to neoplastic melanoma transformation.
RSKs are implicated in the regulation of a wide variety of cellular processes, including gene expression, proliferation, and survival. In humans, mutations in the RSK2 isoform are associated with Coffin-Lowry syndrome, a disease characterized by mental retardation and skeletal abnormalities (12)
. However, the role of RSKs in human cancers has not been established. Our results demonstrate that RSK is both overexpressed and hyperactivated in melanoma cell lines relative to normal melanocytes (Fig. 1B)
. To our knowledge, this is the first demonstration of enhanced RSK expression and activation in tumor cells. Additional studies may help elucidate whether RSK expressed in melanoma cells harbors activating mutations like those found in Ras and B-Raf, or whether its activation is directly attributable to activating mutations in upstream Ras or B-Raf.
RSK specifically phosphorylates Bad at Ser75 in a MAPK-dependent manner (22
, 24
, 42, 43, 44)
, facilitating its inactivation through binding to 14-3-3 and sequestration from heterodimerizing with mitochondria-bound Bcl-2 or Bcl-XL. We demonstrate that Bad Ser75/Ser99/Ser118 phosphorylations occur in a RSK-independent fashion in normal melanocytes (Fig. 2)
, indicating that other survival kinases may be responsible for Bad phosphorylation (44
, 48, 49, 50)
. Treating normal melanocytes with LY294002 or rapamycin, the inhibitors targeting Bad Ser99 kinases Akt and/or p70S6K, fails to inhibit any Bad phosphorylation or induce apoptosis,2
suggesting that neither Akt nor p70S6K functions in Bad-associated melanocyte survival. It remains to be determined whether PKA and/or other survival kinases such as protein kinase C and p21-activated kinase function in Bad-associated cell survival in melanocytes, or, alternatively, whether Bad phosphorylation is associated with melanocyte survival signaling.
Although Bad is highly phosphorylated at Ser75, Bad phosphorylations at Ser99 and Ser118 are low or undetectable in six of seven melanoma cell lines when compared with melanocytes (Fig. 1E)
, and those low-level Bad Ser99/Ser118 phosphorylations are independent of MAPK/RSK activity (Fig. 2, B and C)
. Although inhibiting Bad Ser75 phosphorylation through MEK inhibition induces efficient apoptosis in many melanoma cell lines tested, these cell lines still display differential sensitivities (Fig. 1A
; Ref. 9
). Therefore, although Bad Ser75 phosphorylation is necessary to inhibit the proapoptotic effects of Bad, it alone may not be sufficient to mediate Bad inactivation and melanoma cell survival. Furthermore, we cannot currently rule out the possibility that Bad Ser99/Ser118 phosphorylation, although low, may contribute to cell survival in some melanoma cells. Alternatively, other signaling pathways may couple with Bad inactivation through Ser75 phosphorylation to further regulate melanoma cell survival.
Although a plethora of data exists documenting the proapoptotic role of Bad, only recently have the consequences of Bad Ser75/Ser99/Ser118 phosphorylation been examined in vivo. Using a knock-in approach, Datta et al. (51)
generated a Bad S3A mouse in which all three regulatory serines were substituted with alanines, therein preventing phosphorylation by survival kinases. Although this mouse was viable, suggesting that elements of survival signaling are still functional in the presence of Bad S3A, subsequent analysis documented defects in developmentally regulated apoptosis in certain cell types from the nervous and immune systems. Hence, although insufficient to induce general cell death during development, Bad dephosphorylation is necessary to regulate apoptosis in certain tissues, potentially through collaboration with additional proapoptotic signaling molecules. To this end, expressing CA FOXO3A, a Forkhead transcriptional factor inducing several genes promoting cell death, dramatically enhanced apoptotic response in cerebellar granule neurons derived from Bad S3A mice, suggesting that BadS3A sensitizes cells to the effects of certain death-inducing stimuli in vivo. Furthermore, Bad S3A sensitization was demonstrated to alter the threshold at which mitochondria release cytochrome c in response to apoptotic stimuli. The notion that Bad Ser75/Ser99/Ser118 dephosphorylation is necessary yet not sufficient to induce apoptosis in certain cell types in vivo may suggest a critical, but not exclusive, role for Bad phosphorylation in promoting melanoma cell survival in vitro (Fig. 5)
.
Our results suggest that the MAPK pathway mediates melanoma-specific survival signaling by differentially regulating RSK-mediated phosphorylation of the proapoptotic protein Bad, but not in normal melanocytes. Therefore, interfering with the MEK/ERK/RSK signaling and selectively activating proapoptotic mediators may represent a potential therapeutic strategy in the treatment of melanoma.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Request for reprints: Han-Mo Koo, Laboratory of Cancer Pharmacogenetics, Van Andel Research Institute, 333 Bostwick Avenue, North East, Grand Rapids, Michigan 49503. Phone: (616) 234-5332; Fax: (616) 234-5333; E-mail: hanmo.koo{at}vai.org
1 The abbreviations used are: MAPK, mitogen-activated protein kinase; RSK, ribosomal S6 kinase; MEK, MAPK kinase; ERK, extracellular signal-regulated kinase; RT-PCR, reverse transcription-PCR; GFP, green fluorescent protein; PARP, poly(ADP-ribose) polymerase; BH, Bcl-2 homology; FBS, fetal bovine serum; Wt, wild type; CA, constitutively activated; KD, kinase-dead; VSV-G, vesicular stomatitis virus G; IRES, internal ribosomal entry site; GST, glutathione S-transferase; NHEM, neonatal human epidermal melanocyte; PKA, protein kinase A (cAMP-dependent protein kinase); RT, reverse transcription. 
Received 7/ 1/03. Revised 9/12/03. Accepted 9/23/03.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 L. Chin, L. A. Garraway, and D. E. Fisher Malignant melanoma: genetics and therapeutics in the genomic era. Genes & Dev., August 15, 2006; 20(16): 2149 - 2182. [Abstract] [Full Text] [PDF]
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W. Jia, V. L. Hegde, N. P. Singh, D. Sisco, S. Grant, M. Nagarkatti, and P. S. Nagarkatti {Delta}9-Tetrahydrocannabinol-Induced Apoptosis in Jurkat Leukemia T Cells Is Regulated by Translocation of Bad to Mitochondria Mol. Cancer Res., August 1, 2006; 4(8): 549 - 562. [Abstract] [Full Text] [PDF]
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 K. N. McFarland, S. R. Wilkes, S. E. Koss, K. S. Ravichandran, and J. W. Mandell Neural-specific inactivation of ShcA results in increased embryonic neural progenitor apoptosis and microencephaly. J. Neurosci., July 26, 2006; 26(30): 7885 - 7897. [Abstract] [Full Text] [PDF]
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 U. K. Misra, R. Deedwania, and S. V. Pizzo Activation and Cross-talk between Akt, NF-{kappa}B, and Unfolded Protein Response Signaling in 1-LN Prostate Cancer Cells Consequent to Ligation of Cell Surface-associated GRP78 J. Biol. Chem., May 12, 2006; 281(19): 13694 - 13707. [Abstract] [Full Text] [PDF]
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D. J. Panka, M. B. Atkins, and J. W. Mier Targeting the mitogen-activated protein kinase pathway in the treatment of malignant melanoma. Clin. Cancer Res., April 1, 2006; 12(7): 2371s - 2375s. [Abstract] [Full Text] [PDF]
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D. J. Panka, W. Wang, M. B. Atkins, and J. W. Mier The Raf Inhibitor BAY 43-9006 (Sorafenib) Induces Caspase-Independent Apoptosis in Melanoma Cells Cancer Res., February 1, 2006; 66(3): 1611 - 1619. [Abstract] [Full Text] [PDF]
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S. S. Sridhar, D. Hedley, and L. L. Siu Raf kinase as a target for anticancer therapeutics Mol. Cancer Ther., April 1, 2005; 4(4): 677 - 685. [Abstract] [Full Text] [PDF]
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 M. F. McCarty Targeting Multiple Signaling Pathways as a Strategy for Managing Prostate Cancer: Multifocal Signal Modulation Therapy Integr Cancer Ther, December 1, 2004; 3(4): 349 - 380. [Abstract] [PDF]
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 B. Zhang, Y. Zhang, and E. Shacter Rac1 Inhibits Apoptosis in Human Lymphoma Cells by Stimulating Bad Phosphorylation on Ser-75 Mol. Cell. Biol., July 15, 2004; 24(14): 6205 - 6214. [Abstract] [Full Text] [PDF]
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