Selective Activation of Mitogen-Activated Protein (MAP) Kinase Kinase 3 and p38α MAP Kinase Is Essential for Cyclic AMP-Dependent UCP1 Expression in Adipocytes

Abstract

The sympathetic nervous system regulates the activity and expression of uncoupling protein 1 (UCP1) through the three β-adrenergic receptor subtypes and their ability to raise intracellular cyclic AMP (cAMP) levels. Unexpectedly, we recently discovered that the cAMP-dependent regulation of multiple genes in brown adipocytes, including Ucp1, occurred through the p38 mitogen-activated protein kinases (MAPK) (W. Cao, K. W. Daniel, J. Robidoux, P. Puigserver, A. V. Medvedev, X. Bai, L. M. Floering, B. M. Spiegelman, and S. Collins, Mol. Cell. Biol. 24:3057-3067, 2004). However, no well-defined pathway linking cAMP accumulation or cAMP-dependent protein kinase (PKA) to p38 MAPK has been described. Therefore, in the present study using both in vivo and in vitro models, we have initiated a retrograde approach to define the required components, beginning with the p38 MAPK isoforms themselves and the MAP kinase kinase(s) that regulates them. Our strategy included ectopic expression of wild-type and mutant kinases as well as targeted inhibition of gene expression using small interfering RNA. The results indicate that the β-adrenergic receptors and PKA lead to a highly selective activation of the p38α isoform of MAPK, which in turn promotes Ucp1 gene transcription. In addition, this specific activation of p38α relies solely on the presence of MAP kinase kinase 3, despite the expression in brown fat of MKK3, -4, and -6. Finally, of the three scaffold proteins of the JIP family expressed in brown adipocytes, only JIP2 coimmunoprecipitates p38α MAPK and MKK3. Therefore, in the brown adipocyte the recently described scaffold protein JIP2 assembles the required factors MKK3 and p38α MAPK linking PKA to the control of thermogenic gene expression.

ACKNOWLEDGMENTS

We thank Leslie P. Kozak, Jiahuai Han, Roger J. Davis, Josef M. Penninger, and E. Premkumar Reddy for their invaluable gifts of plasmids (listed in Materials and Methods). We also thank Alexander Medvedev for helpful discussions in the initial phase of the project.

This work was supported by NIH awards R01 DK57698 and R01 DK53092 (S.C.) and a fellowship from Fonds de la Recherche en Santé du Québec (J.R.).