G1 ARREST FOLLOWS LOSS OF CYTOKINE SIGNALING THROUGH DEGRADATION OF Cdc25A, MEDIATED BY p38 MAP KINASE

Annette R. Khaled^1,3 *, Amy Grenier¹, Michelle Alvarez¹, Dmitry V. Bulavin², Wen Qing Li³, Kyungjae Kim^3,4, Albert J. Fornace², and Scott K. Durum³ 

¹University of Central Florida, BioMolecular Science Center, Orlando, FL, 32628 ²Division of Basic Sciences, NCI, Bethesda, MD, 20892, ³Laboratory of Molecular Immunoregulation, NCI-Frederick, Frederick, MD, 21702, ⁴Dept. of  Pharmacy, Sahm-Yook University, Seoul, Korea, 139-742.    *akhaled@mail.ucf.edu

INTRODUCTION.  Survival and growth of lymphocytes both result from signals transduced through cytokine receptors.  Interleukin-7 (IL-7) and interleukin-3 (IL-3) are essential cytokines that promote cell survival by regulating the balance of apoptotic mediators¹. However, a cytokine signal is also necessary and required for cell cycle progression². Enzymatic complexes containing cyclin-dependent kinases (cdks) and cyclins catalyze cell cycling.  The Cell division cycle 25 (Cdc25) family of phosphatases activates cdks through dephosphorylation of inhibitory sites.  To dissect the critical signaling events that produce growth arrest in the absence of cytokine signaling, we studied IL-3- and IL-7-dependent cell lines to identify those components most important for cytokine-mediated cell cycle control.

METHOD.  Two cytokine-dependent cell lines were used: the IL-3 dependent pro-B cell line, FL5.12A, and the IL-7-dependent thymic line, D1 ³. Cells were treated with pharmacological inhibitors of the MAP kinases (MAPK), or transfected with dominant negative MAPKs, to block kinase activity during cytokine withdrawal.  The pCMV-HA-Cdc25 WT plasmid was used to generate mutants pCMV-HA-Cdc25, S75A and S123A⁴. Cells were transfected with plasmids by electroporation. Protein levels of Cdc25A WT and mutants were assayed by immunoblotting.  Cell cycling was assayed by propidium iodide (PI) staining and analyzed by flow cytometry.  Incorporation of BrdU was used to assess DNA synthesis.

RESULTS.  Withdrawal of IL-3 or IL-7 progressively induced G₁ arrest in FL5.12A or D1 cells.  To determine how the absence of a cytokine signal induced growth arrest, we inhibited the activity of the MAP kinase family, using pharmacological inhibitors or dominant negative kinases.  Only inhibition of p38 MAP kinase (p38) induced S-phase progression in FL5.12A or D1 cells deprived of cytokines.  Inhibition of other MAP kinases, ERK or JNK, had no effect.  Overexpression of Cyclin D1 did not promote cell cycling in the absence of cytokines.  However, since inhibition of Cdk2 countered the effect of p38 inhibition following IL-3 withdrawal, we examined the phosphatase, Cdc25A, known to remove the inhibitory phosphorylation from Cdk2.  We observed that Cdc25A rapidly degraded following cytokine withdrawal and was subsequently restored by inhibiting p38.  Two critical serines on Cdc25A, S75 and S123, were shown to be targets of p38 phosphorylation by in vitro kinase assay.  Mutation of both of these residues significantly restored S-phase entry following IL-7 or IL-3 withdrawal, even in the presence of the negative regulator, p27^kip1.  Like inhibition of p38, mutation of S75 and S123 stabilized the Cdc25A protein following cytokine deprivation, resulting in the accumulation of activated phospho-Cdk2, phospho-retinoblastoma protein and renewed cell cycling.

DISCUSSION:  We show that control of G1-S phase progression through cytokines is centrally mediated by the phosphatase, Cdc25A.  Stability of Cdc25A is, in turn, regulated by p38.  In a recent study, HeLa cells, expressing the stable Cdc25A mutein, still arrested when exposed to osmotic stress or UV irradiation ⁴.  These findings suggest that induction of G₁ arrest in lymphocytes during cytokine withdrawal is solely modulated through Cdc25A, whereas growth arrest through stress, as shown with HeLa cells, triggers additional regulatory mechanisms. The implications of the presented findings are significant, demonstrating that regulation of cell cycling through Cdc25A is a critical function of cytokines in their roles as mediators of lymphocyte development and peripheral homeostasis.

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