Inhibition of Cell Proliferation and Cell Cycle Progression by Specific Inhibition of Basal JNK Activity: EVIDENCE THAT MITOTIC Bcl-2 PHOSPHORYLATION IS JNK-INDEPENDENT

doi:10.1074/jbc.M304935200

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Inhibition of Cell Proliferation and Cell Cycle Progression by Specific Inhibition of Basal JNK Activity

EVIDENCE THAT MITOTIC Bcl-2 PHOSPHORYLATION IS JNK-INDEPENDENT^*

Lihua Du ${ddagger}$ $§$ , Christopher S. Lyle ${ddagger}$ $§$ , Toria B. Obey ${ddagger}$ , William A. Gaarde¶, Jeffrey A. Muir||, Brydon L. Bennett||, and Timothy C. Chambers ${ddagger}$ **

From the Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, ^¶ISIS Pharmaceuticals, Inc., Carlsbad, California 92008, and the ^||Signal Research Division, Celgene, San Diego, California 92121

The c-Jun NH₂-terminal kinase (JNK) subgroup of mitogen-activatedprotein kinases has been implicated largely in stress responses,but an increasing body of evidence has suggested that JNK alsoplays a role in cell proliferation and survival. We examinedthe effect of JNK inhibition, using either SP600125 or specificantisense oligonucleotides, on cell proliferation and cell cycleprogression. SP600125 was selective for JNK in vitro and invivo versus other kinases tested including ERK, p38, cyclin-dependentprotein kinase 1 (CDK1), and CDK2. SP600125 inhibited JNK activityand KB-3 cell proliferation with the same dose dependence, suggestingthat inhibition of proliferation was a direct consequence ofJNK inhibition. Inhibition of proliferation by SP600125 wasassociated with an increase in the G₂-M and apoptotic fractionsof cells but was not associated with p53 or p21 induction. Antisenseoligonucleotides to JNK2 but not JNK1 caused highly significantinhibition of cell proliferation. Wild-type mouse fibroblastsresponded similarly with proliferation inhibition and apoptosisinduction, whereas c-jun^-/- fibroblasts were refractory to theeffects of SP600125, suggesting that JNK signaling to c-Junis required for cell proliferation. Studies in synchronizedKB-3 cells indicated that SP600125 delayed transit time throughS and G₂-M phases. Correspondingly, JNK activity increased inlate S phase and peaked in late G₂ phase. During synchronousmitotic progression, cyclin B levels increased concomitant withphosphorylation of c-Jun, H1 histone, and Bcl-2. In the presenceof SP600125, mitotic progression was prolonged, and c-Jun phosphorylationwas inhibited, but neither H1 nor Bcl-2 phosphorylation wasinhibited. However, the CDK inhibitor roscovitine inhibitedmitotic Bcl-2 phosphorylation. These results indicate that JNK,and more specifically the JNK2 isoform, plays a key role incell proliferation and cell cycle progression. In addition,conclusive evidence is presented that a kinase other than JNK,most likely CDK1 or a CDK1-regulated kinase, is responsiblefor mitotic Bcl-2 phosphorylation.

Received for publication, May 12, 2003 , and in revised form, December 26, 2003.

^* This work was supported by National Institutes of Health GrantCA 75577. The costs of publication of this article were defrayedin part by the payment of page charges. This article must thereforebe hereby marked "advertisement" in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

^** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Mail Slot 516, 4301 W. Markham St., Little Rock, AR 72205-7199. E-mail: chamberstimothyc{at}uams.edu.

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