The mitogen-activated protein kinase JNK1 suppresses interleukin-3 withdrawal-induced cell death through phosphorylation of the BH3-only pro-apoptotic Bcl-2 family protein Bad at Thr-201. It is unknown whether JNK1 regulates glycolysis, an important metabolic process that is involved in cell survival, and if so, whether the regulation depends on Thr-201 phosphorylation of Bad. Here we report that phosphorylation of Bad by JNK1 is required for glycolysis through activation of phosphofructokinase-1 (PFK-1), one of the key enzymes that catalyze glycolysis. Genetic disruption of Jnk1 alleles or silencing of Jnk1 by small interfering RNA abrogates glycolysis induced by growth/survival factors such as serum or interleukin-3. Proteomic analysis identifies PFK-1 as a novel Bad-associated protein. Although the interaction between PFK-1 and Bad is independent of JNK1, Thr-201 phosphorylation of Bad by JNK1 is required for PFK-1 activation. Thus, our results provide a novel molecular mechanism by which JNK1 promotes glycolysis for cell survival.The c-Jun N-terminal protein kinase (JNK; 2 also known as stress-activated protein kinase, SAPK) (1), a subfamily of the mitogen-activated protein kinase (MAPK) superfamily, has two ubiquitously expressed isoforms, JNK1 and JNK2, and a tissue-specific isoform JNK3 with different splicing variant (2-4). Between JNK1 and JNK2, JNK1 is the main c-Jun kinase (5-7). JNK is activated by a variety of extracellular stimuli, including growth factors, cytokines, oncogenes, and environmental stresses through a MAP kinase module, i.e. MAP3K 3 MAP2K 3 MAPK (4). Two MAP2Ks (JNKK1/ MKK4/SEK1 and JNKK2/MKK7) for JNK have been identified (8 -11). Several MAP3Ks, such as MEKK1 (12), MEKK2 (13), apoptosis signal-regulating kinase (ASK1) (14), mixed lineage kinase (MLK) (15), TGF-activated kinase (TAK1) (16), tumor repression locus-2 (Tpl-2) (17), dual leucine zipper-bearing kinase (DLK) (18), and thousand and one amino acid kinase (Tao) (19), have been reported to act as MAP3Ks for JNK. Activation of JNK is also regulated by scaffold proteins such as JIP, -arrestin, JSAP1 (20 -22), and protein phosphatases such as mitogen-activated protein phosphatases (23). In addition, the transcription factor NF-B can negatively or positively regulate JNK activation, depending on the nature of the stimulus (7, 24 -29). JNK plays a critical role in many cellular activities, from growth control to programmed cell death, and deregulation of JNK activity has been implicated in many human diseases and certain types of cancer (28, 30 -33).Bad is a BH3-only pro-apoptotic Bcl-2 family protein, having two splicing forms Bad s and Bad L (34). Bad plays a critical role in the cross-talk between the growth/survival factor signaling pathway and the intrinsic death machinery (35). The pro-apoptotic activity of Bad is regulated by extracellular stimuli via post-translational modifications including phosphorylation (35)(36)(37)(38). In response to the stimulation of growth/survival factors such as interleukin-3 (IL-3) and insul...