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Interleukin (IL)-6 is a pleiotropic hormone that has both proinflammatory and anti-inflammatory actions. AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that among its other actions responds to decreases in cellular energy state by enhancing processes that generate ATP and inhibiting others that consume ATP but are not acutely necessary for survival. IL-6 is synthesized and released from skeletal muscle in large amounts during exercise, and in rodents, the resultant increase in its concentration correlates temporally with increases in AMPK activity in multiple tissues. That IL-6 may be responsible in great measure for these increases in AMPK is suggested by the fact it increases AMPK activity both in muscle and adipose tissue in vivo and in incubated muscles and cultured adipocytes. In addition, we have found that AMPK activity is diminished in muscle and adipose tissue of 3-month-old IL-6 knockout (KO) mice at rest and that the absolute increases in AMPK activity in these tissues caused by exercise is diminished compared with control mice. Except for an impaired ability to exercise and to oxidize fatty acids, the IL-6 KO mouse appears normal at 3 months of age. On the other hand, by age 9 months, it manifests many of the abnormalities of the metabolic syndrome including obesity, dyslipidemia, and impaired glucose tolerance. This, plus the association of decreased AMPK activity with similar abnormalities in a number of other rodents, suggests that a decrease in AMPK activity may be a causal factor. Whether increases in IL-6, by virtue of their effects on AMPK, contribute to the reported ability of exercise to diminish the prevalence of type 2 diabetes, coronary heart disease, and other disorders associated with the metabolic syndrome remains to be determined. Diabetes 55 (Suppl. 2):S48 -S54, 2006

Growth factors activate Raf-1 by engaging a complex program, which requires Ras binding, membrane recruitment, and phosphorylation of Raf-1. The present study employs the microtubule-depolymerizing drug nocodazole as an alternative approach to explore the mechanisms of Raf activation. Incubation of cells with nocodazole leads to activation of Pak1/2, kinases downstream of small GTPases Rac/Cdc42, which have been previously indicated to phosphorylate Raf-1 Ser 338 . Nocodazole-induced stimulation of Raf-1 is augmented by co-expression of small GTPases Rac/Cdc42 and Pak1/2. Dominant negative mutants of these proteins block activation of Raf-1 by nocodazole, but not by epidermal growth factor (EGF). Thus, our studies define Rac/ Cdc42/Pak as a module upstream of Raf-1 during its activation by microtubule disruption. Although it is Ras-independent, nocodazole-induced activation of Raf-1 appears to involve the amino-terminal regulatory region in which the integrity of the Ras binding domain is required. Surprisingly, the Raf zinc finger mutation (C165S/C168S) causes a robust activation of Raf-1 by nocodazole, whereas it diminishes Ras-dependent activation of Raf-1. We also show that mutation of residues Ser 338 to Ala or Tyr 340 -Tyr 341 to Phe-Phe immediately amino-terminal to the catalytic domain abrogates activation of both the wild type and zinc finger mutant Raf by both EGF/4␤-12-O-tetradecanoylphorbol-13-acetate and nocodazole. Finally, an in vitro kinase assay demonstrates that the zinc finger mutant serves as a better substrate of Pak1 than the wild type Raf-1. Collectively, our results indicate that 1) the zinc finger exerts an inhibitory effect on Raf-1 activation, probably by preventing phosphorylation of 338 SSYY 341 ; 2) such inhibition is first overcome by an unknown factor binding in place of Ras-GTP to the amino-terminal regulatory region in response to nocodazole; and 3) EGF and nocodazole utilize different kinases to phosphorylate Ser 338 , an event crucial for Raf activation.The proto-oncogene raf-1, first identified as a cellular counterpart of the oncogene v-raf, encodes a serine/threonine protein kinase. Raf-1 is ubiquitously expressed and plays an important role in cell proliferation and differentiation (1). The mechanism by which Raf-1 is activated by growth factors is still incompletely understood, although it is known to be preassembled as a complex with 14-3-3 and heat shock proteins 90/50 (2-6). It involves multiple steps including Ras-GTP binding, membrane recruitment, and phosphorylation.Raf-1 consists of an amino-terminal regulatory domain and a carboxyl-terminal kinase domain. The amino-terminal moiety of Raf-1 exerts an inhibitory effect on the catalytic activity, since amino-terminal truncations lead to progressive increases in its transforming ability (7,8). The amino-terminal regulatory region of Raf-1 contains a Ras binding domain (RBD) 1 and a cysteine-rich zinc finger domain (CRD), both of which participate in binding to Ras. The first interaction engages Raf RBD ranging from ...