Inhibition of Calcium/Calmodulin-dependent Protein Kinase Kinase by Protein 14-3-3

• Published in: The Journal of biological chemistry 2004-12, Vol.279 (50), p.52191-52199
• Main Authors: Davare, Monika A, Saneyoshi, Takeo, Guire, Eric S, Nygaard, Sean C, Soderling, Thomas R
• Format: Article
• Language: English
• Subjects: 14-3-3 Proteins - genetics; 14-3-3 Proteins - metabolism; 14-3-3 Proteins - pharmacology; Animals; Binding Sites; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Cell Line; Colforsin - pharmacology; COS Cells; Cyclic AMP-Dependent Protein Kinases - antagonists & inhibitors; Cyclic AMP-Dependent Protein Kinases - metabolism; Enzyme Inhibitors - pharmacology; Hippocampus - drug effects; Hippocampus - metabolism; Humans; In Vitro Techniques; Phosphorylation; Protein-Serine-Threonine Kinases - antagonists & inhibitors; Protein-Serine-Threonine Kinases - chemistry; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Rats; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacology; Signal Transduction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M409873200

Intracellular calcium concentrations regulate diverse cellular events including cytoskeletal dynamics, gene transcription, and synaptic plasticity. The calcium signal is transduced in part by the calcium/calmodulin-dependent protein kinase (CaMK) cascade that is comprised of CaMK kinase (CaMKK) and its primary downstream substrates, CaMKI and CaMKIV. The CaMK cascade also participates in cross-talk with other signaling pathways: CaMKK/CaMKI can activate the mitogen-activated protein kinase pathway and cAMP-dependent protein kinase (PKA) can directly phosphorylate two inhibitory sites (Thr 108 and Ser 458 ) in CaMKK. Here we report an additional PKA-dependent regulation of CaMKK through its interaction with protein 14-3-3. CaMKK and 14-3-3 co-immunoprecipitated from co-transfected heterologous cells as well as from rat brain homogenate, and site-directed mutagenesis studies identified phospho-Ser 74 in CaMKK as the primary 14-3-3 binding site. In cultured rat hippocampal neurons and acute hippocampal slices this interaction was robustly stimulated by activation of PKA through forskolin treatment and was blocked by inhibition of PKA. Interaction of 14-3-3 with CaMKK had two regulatory consequences in vitro . It directly inhibited CaMKK activity, and it also blocked dephosphorylation of Thr 108 , an inhibitory PKA phosphorylation site. In human embryonic kidney 293 cells transfected with CaMKK and stimulated with forskolin, co-transfection with 14-3-3 prevented dephosphorylation of Thr 108 to the same extent as did inhibition of protein phosphatases with okadaic acid. We conclude that binding of 14-3-3 to CaMKK stabilizes its inhibition by PKA-mediated phosphorylation, which may have important consequences in the regulation of CaMKI, CaMKIV, protein kinase B, and ERK signaling pathways.