Constitutive expression of the Pim-1 kinase prolongs survival of cytokine-deprived FDCP1 cells, partly via maintenance of Bcl-2 expression. Here, we show that Pim-1 colocalizes and physically interacts with the pro-apoptotic Bad protein and phosphorylates it in vitro on serine 112, which is a gatekeeper site for its inactivation. Furthermore, wild-type Pim-1, but not a kinase-deficient mutant, enhances phosphorylation of this site in FDCP1 cells and protects cells from the pro-apoptotic effects of Bad. Our results suggest that phosphorylation of Bad by Pim-1 is one of several mechanisms via which the Pim-1 kinase can enhance Bcl-2 activity and promote cell survival.

IntroductionInterleukin-3 (IL-3) induces growth and differentiation of several types of cell populations, including multipotential hematopoietic stem cells, immature myeloid cells, as well as T-and pre-Blymphoid cells. The IL-3 receptor along with the related receptors for IL-5 and granulocyte macrophage-colony-stimulating factor (GM-CSF) are composed of a ligand-binding alpha chain and a signal-transducing beta chain, which heterodimerize upon ligand binding. 1 The membrane-proximal intracellular region of the beta chain is required for induction of proliferation-associated genes such as c-myc and pim-1. 2,3 This region is also bound by multiple signal-transducing proteins such as Janus kinases (JAKs), signal transducers and activators of transcription (STATs), c-Src, and phosphotidylinositol-3 kinase. 4 STAT5 with its 2 isoforms, STAT5A and STAT5B, plays a critical role in proliferation of hematopoietic cells in response to IL-2, IL-3, IL-5, GM-CSF, erythropoietin (EPO), prolactin (PRL), epidermal growth factor (EGF), and growth hormone. [5][6][7] Activation of STAT5 by the oncogenic Bcr/Abl fusion protein is essential for its ability to transform hematopoietic cells, 8 and STAT5 has been implicated in proliferation and survival of several types of cancer cells. 9 Moreover, mutationally activated forms of STAT5 have been demonstrated to possess transforming capacity. 10,11 After cytokine stimulation, STAT5 is phosphorylated on tyrosine by JAK2, dimerized, and translocated to the nucleus, where it induces expression of numerous proteins including cyclin D, Bcl-x L , Pim-1, c-Fos, c-Jun, and suppressors of cytokine signaling such as cytokine-inducible SH2-containing protein (CIS) and suppressor of cytokine signaling (SOCS) proteins. 12-16 STAT5 is also regulated by serine phosphorylation by both mitogen-activated protein kinase (MAPK)-dependent and -independent pathways, 17 but the exact mechanisms and kinases involved remain to be identified.The pim-1 oncogene was originally identified as a common integration site for Moloney murine leukemia virus, 18 and was shown to efficiently cooperate with c-myc, N-myc, or bcl-2 genes in lymphomagenesis. 19,20 In the mouse, there are 2 major isoforms of the serine/threonine-specific Pim-1 kinase, 33 kDa and 44 kDa, the larger one being initiated from an upstream alternative translational initiation site. 21 The expression of pim-1 is induced by several related cytokines including IL-2, IL-3, GM-CSF, EPO, and PRL, 3,5,22,23 all of which also activate STAT5. Indeed, pim-1 is a STAT5 target gene. Moreover, it has been proposed that the IL-3 response mediated by STAT5 may be processed by the products of the STAT5 target genes c-fos and pim-1. 12,16 More recently, pim-1 was shown to be up-regulated by Bcr/Abl via STAT5 and be required for Bcr/Abl-mediated cell survival and transformation. 24 Indeed, Pim-1 can protect hematopoietic cells from apoptosis induced by cytokine withdrawal, 25 glucocorticoids, 26 or genotoxic stress. 27 In addition, Pim-1 can stimulate activities of se...