title: Analysis of a cAMP regulated coactivator family reveals an alternative phosphorylation motif for AMPK family members.
abstract: The second messenger cAMP stimulates cellular gene expression via the PKA-mediated phosphorylation of the transcription factor CREB and through dephosphorylation of the cAMP-responsive transcriptional coactivators (CRTCs). Under basal conditions, CRTCs are phosphorylated by members of the AMPK family of Ser/Thr kinases and sequestered in the cytoplasm via a phosphorylation-dependent association with 14-3-3 proteins. Increases in cAMP promote the dephosphorylation and nuclear translocation of CRTCs, where they bind to CREB and stimulate relevant target genes. Although they share considerable sequence homology, members of the CRTC family exert non-overlapping effects on cellular gene expression through as yet unidentified mechanisms. Here we show that the three CRTCs exhibit distinct patterns of 14-3-3 binding at three conserved sites corresponding to S70, S171, and S275 (in CRTC2). S171 functions as the gatekeeper site for 14-3-3 binding; it acts cooperatively with S275 in stabilizing this interaction following its phosphorylation by the cAMP-responsive SIK and the cAMP-nonresponsive MARK kinases. Although S171 contains a consensus recognition site for phosphorylation by AMPK family members, S70 and S275 carry variant motifs (MNTGGS275LPDL), lacking basic residues that are otherwise critical for SIK/MARK recognition as well as 14-3-3 binding. Correspondingly, the activity of these motifs differs between CRTC family members. As the variant (SLPDL) motif is present and apparently phosphorylated in other mammalian proteins, our studies suggest that the regulation of cellular targets by AMPK family members is more extensive than previously appreciated.
authors.affiliation: Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California, United States of America.
authors.forename: Tim
authors.identifier: http://orcid.org/0000-0001-8487-6963
authors.initials: T
authors.lastname: Sonntag
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, United States of America.
authors.forename: James J
authors.initials: JJ
authors.lastname: Moresco
authors.affiliation: Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California, United States of America.
authors.forename: Joan M
authors.initials: JM
authors.lastname: Vaughan
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto, Japan.
authors.forename: Shigenobu
authors.initials: S
authors.lastname: Matsumura
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, United States of America.
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.affiliation: Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California, United States of America.
authors.forename: Marc
authors.initials: M
authors.lastname: Montminy
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000378:metabolism; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011817:Rabbits; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D048948:14-3-3 Proteins; C555318:CRTC3 protein, mouse; C519509:Crtc1 protein, mouse; C504923:Crtc2 protein, mouse; D014157:Transcription Factors; D000242:Cyclic AMP; D017346:Protein Serine-Threonine Kinases