Insulin disruptsb-adrenergic signalling to proteinkinase A in adipocytesJin Zhang1*†, Christopher J. Hupfeld2*, Susan S. Taylor3,4, Jerrold M. Olefsky2& Roger Y. Tsien1,3,4Hormones mobilize intracellular second messengers and initiatesignalling cascades involving protein kinases and phosphatases,which are often spatially compartmentalized by anchoring pro-teins to increase signalling specificity1. These scaffold proteinsmay themselves be modulated by hormones2–4. In adipocytes,stimulation ofb-adrenergic receptors increases cyclic AMP levelsand activates protein kinase A (PKA)5, which stimulates lipolysisby phosphorylating hormone-sensitive lipase and perilipin6–8.Acute insulin treatment activates phosphodiesterase 3B, reducescAMP levels and quenchesb-adrenergic receptor sig nalling9.Incontrast, chronic hyperinsulinaemic conditions (typical of type 2diabetes) enhanceb-adrenergic receptor-mediated cAMP pro-duction10. This amplification of cAMP signalling is paradoxicalbecause it should enhance lipolysis, the opposite of the knownshort-term effect of hyperinsulinaemia. Here we show that inadipocy tes, chronically high insulin levels inhibitb-adrenergicreceptors (but not other cAMP-elevating stimuli) from activatingPKA. We measured this using an improved fluorescent reporterand by phosphorylation of endogenous cAMP-response-elementbinding protein (CREB). Disruption of PKA scaffolding mimicsthe interference of insulin withb-adrenergic receptor signalling.Chronically high insulin levels may disrupt the close apposition ofb-adrenergic receptors and PKA, identifying a new mechanism forcrosstalk between heterologous signal transduction pathways.In order to assess the effect of chronically high insulin levels onPKA activation (a signalling step immediately following cAMPproduction), we took advantage of genetically encoded A-kinaseactivity reporters (AKARs) for monitoring PKA activity in livingcells. Such reporters serve as surrogate substrates for PKA and, whenphosphorylated, generate a change in fluorescence resonance energytransfer (FRET) between two green fluorescent protein (GFP)mutants as the result of phosphorylation-induced intramolecularcomplex formation between a phosphoamino acid binding domainand the phosphorylated peptide11. The previously described AKAR1reporter has successfully monitored compartmentalized PKAactivity. However, its poor sensitivity to cellular phosphatasesimpedes reversal of the FRET response, limiting the utility ofAKAR1 for physiological studies.To generate an improved AKAR reporter, we hypothesized thatwithin the AKAR construct, tight binding of the phosphorylatedpeptide by the 14-3-3 module efficiently protects it from dephos-phorylation12. Thus, a phosphoamino acid binding domain that hasreduced binding affinity for the phosphorylated peptide would bemore suitable for constructing a reversible reporter. To this end,we chose the forkhead associated domain 1 (FHA1), a modularphosphothreonine binding domain with submicromolar bindingaffinity13, one order of magnitude weaker than that of 14-3-3 andsubstrate peptides. Furthermore, peptide library screening hasidentified FHA1-binding peptides that favour specific amino acidsaround the phosphorylation site, particularly in the positionsimmediately C-terminal to the phospho-threonine (pT)13.Wetherefore modified the PKA substrate sequence to LRRATLVD byincorporating the near-optimal sequence for FHA1 binding atpositions þ1, þ 2 and þ3 with respect to pT, while maintainingthe PKA phosphorylation consensus motif at the 22, 23 positions(Fig. 1a).We incorporated three linkers of different lengths between thesubstrate sequence and FHA1, and found that one construct pro-vided a greater response and faster kinetics compared to the othertwo constructs (data not shown). This reporter is termed AKAR2,and when tested in HEK293 cells, it showed a reversible response.After the response induced by theb-adrenergic agonist isoproterenolreached a maximum, removal of the stimulant and addition of H89(a relatively specific PKA inhibitor) led to a decrease in emissionratio, which returned to the basal level over 30–50 min (Fig. 1b). Asecond increase in emission ratio was generated by removal of H89and addition of the adenylyl cyclase activator forskolin, showing thatactivation of AKAR2 was fully reversible. Pulsed photolytic release(‘uncaging’) of cAMP from a membrane-permeant precursor14ledto repeated cycles of increased and decreased emission ratios, pre-sumably involving intracellular release then rapid degradation ofcAMP followed by rapid changes in the activity balance between PKAand phosphatases (Fig. 1c).To test the specificity of the reporter, we incubated AKAR2-expressing HEK293 cells with H89. As seen in Fig. 1d, this pretreat-ment prevented the response of AKAR2 to isoproterenol. Thereporter itself was not compromised, as removal of H89 restoredthe ability of AKAR2 to respond to forskolin. Furthermore, co-expression of the specific PKA inhibitor PKI also abolished theresponse (Fig. 1e), confirming that the FRET response is PKA-specific. In addition, treatment of AKAR2-expressing HEK293 cellswith phorbol dibutyrate (PDBu, a protein kinase C (PKC) activator)or thapsigargin (to stimulate calcium/calmodulin-dependent pro-tein kinase II, CaMKII) did not alter the emission ratio (Fig. 1f, g).Thus, AKAR2 senses PKA but not PKC or CaMKII activation inliving cells.To confirm that changes in emission ratio result from phosphoryl-ation of AKAR2 at the designated threonine residue, we changed thethreonine to alanine in the PKA substrate sequence (LRRATLVD).Figure 1h shows that this single mutation, T391A, completelyabolished the response to increases in cAMP. To compare the FRETchange with the phosphorylation state of the reporter, immuno-blotting analysis was performed using an antiphospho-PKA-substrate antibody before and after forskolin stimulation. AKAR2LETTERS1Department of Pharmacology,2Division of Endocrinology and Metabolism,3Department of Chemistry and Biochemistry, and4Howard Hughes Medical Institute, University ofCalifornia at San Diego, La Jolla, California 92093, USA. †Present address: Department of Pharmacology and Molecular Sciences and Department of Neuroscience, The JohnsHopkins University School of Medicine, Baltimore, Maryland 21205, USA.*These authors contributed equally to this work.Vol 437|22 September 2005|doi:10.1038/nature04140569© 2005 Nature Publishing Groupmigrated at the expected