SUMMARY The primary task of white adipose tissue (WAT) is the storage of lipids. However, ‘beige’ adipocytes also exist in WAT. Beige adipocytes burn fat and dissipate the energy as heat, but their abundance is diminished in obesity. Stimulating beige adipocyte development, or WAT browning, increases energy expenditure and holds potential for combating metabolic disease and obesity. Here we report that insulin and leptin act together on hypothalamic neurons to promote WAT browning and weight loss. Deletion of the phosphatases PTP1B and TCPTP enhanced insulin and leptin signaling in proopiomelanocortin neurons and prevented diet-induced obesity by increasing WAT browning and energy expenditure. The co-infusion of insulin plus leptin into the CNS or the activation of proopiomelanocortin neurons also increased WAT browning and decreased adiposity. Our findings identify a homeostatic mechanism for coordinating the status of energy stores, as relayed by insulin and leptin, with the central control of WAT browning.

The dephosphorylation of p-nitrophenyl phosphate by Yersinia protein-tyrosine phosphatase (PTPase) and by the rat PTP1 has been examined by measurement of heavy-atom isotope effects at the nonbridge oxygen atoms [18(V/K)nonbridge], at the bridging oxygen atom [18(V/K)bridge], and the nitrogen atom in the leaving group 15(V/K). The effects were measured using an isotope ratio mass spectrometer by the competitive method and thus are effects on V/K. The results for the Yersinia PTPase and rat PTP1, respectively, are 1.0142 +/- 0.0004 and 1.0152 +/- 0.0006 for 18(V/K)bridge; 0.9981 +/- 0.0015 and 0.9998 +/- 0.0013 for 18(V/K)nonbridge; and 1.0001 +/- 0.0002 and 0.9999 +/- 0.0003 for 15(V/K). The magnitudes of the isotope effects are similar to the intrinsic values measured in solution, indicating that the chemical step is rate-limiting for V/K. The transition state for phosphorylation of the enzyme is dissociative in character, as is the case in solution. Binding of the substrate is rapid and reversible, as is the binding-induced conformational change which brings the catalytic general acid into the active site. Cleavage of the P-O bond and proton transfer from the general acid Asp to the leaving group are both far advanced in the transition state, and there is no development of negative charge on the departing leaving group. Experiments with several general acid mutants give values for 18(V/K)bridge of around 1.0280, 15(V/K) of about 1.002, and 18(V/K)nonbridge effects of from 1.0007 to 1.0022. These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion but suggest more nucleophilic participation than in the solution reaction.

The extracellular signal-regulated protein kinase 2 (ERK2) is the founding member of a family of mitogenactivated protein kinases (MAPKs) that are central components of signal transduction pathways for cell proliferation, stress responses, and differentiation. The MAPKs are unique among the Ser/Thr protein kinases in that they require both Thr and Tyr phosphorylation for full activation. The dual phosphorylation of Thr-183 and Tyr-185 in ERK2 is catalyzed by MAPK/ERK kinase 1 (MEK1). However, the identity and relative activity of protein phosphatases that inactivate ERK2 are less well established. In this study, we performed a kinetic analysis of ERK2 dephosphorylation by protein phosphatases using a continuous spectrophotometric enzymecoupled assay that measures the inorganic phosphate produced in the reaction. Eleven different protein phosphatases, many previously suggested to be involved in ERK2 regulation, were compared, including tyrosinespecific phosphatases (PTP1B, CD45, and HePTP), dual specificity MAPK phosphatases (VHR, MKP3, and MKP5), and Ser/Thr protein phosphatases (PP1, PP2A, PP2B, PP2C␣, and PP). The results provide biochemical evidence that protein phosphatases display exquisite specificity in their substrate recognition and implicate HePTP, MKP3, and PP2A as ERK2 phosphatases. The fact that ERK2 inactivation could be carried out by multiple specific phosphatases shows that signals can be integrated into the pathway at the phosphatase level to determine the cellular response to external stimuli. Important insights into the roles of various protein phosphatases in ERK2 kinase signaling are obtained, and further analysis of the mechanism by which different protein phosphatases recognize and inactivate MAPKs will increase our understanding of how this kinase family is regulated.

Protein-tyrosine phosphatses (PPases)

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