title: cAMP-stimulated phosphorylation of diaphanous 1 regulates protein stability and interaction with binding partners in adrenocortical cells.
abstract: Diaphanous homologue 1 (DIAPH1) is a Rho effector protein that coordinates cellular dynamics by regulating microfilament and microtubule function. We previously showed that DIAPH1 plays an integral role in regulating the production of cortisol by controlling the rate of mitochondrial movement, by which activation of the adrenocorticotropin (ACTH)/cAMP signaling pathway stimulates mitochondrial trafficking and promotes the interaction between RhoA and DIAPH1. In the present study we use mass spectrometry to identify DIAPH1 binding partners and find that DIAPH1 interacts with several proteins, including RhoA, dynamin-1, kinesin, β-tubulin, β-actin, oxysterol-binding protein (OSBP)-related protein 2 (ORP2), and ORP10. Moreover, DIAPH1 is phosphorylated in response to dibutyryl cAMP (Bt2cAMP) at Thr-759 via a pathway that requires extracellular signal-related kinase (ERK). Alanine substitution of Thr-759 renders DIAPH1 more stable and attenuates the interaction between DIAPH1 and kinesin, ORP2, and actin but has no effect on the ability of the protein to interact with RhoA or β-tubulin. Finally, overexpression of a DIAPH1 T759A mutant significantly decreases the rate of Bt2cAMP-stimulated mitochondrial movement. Taken together, our findings establish a key role for phosphorylation in regulating the stability and function of DIAPH1.
authors: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093-0704, USA.
authors: Donghui
authors: D
authors: Li
authors: Eric B
authors: EB
authors: Dammer
authors: Natasha C
authors: NC
authors: Lucki
authors: Marion B
authors: MB
authors: Sewer
mesh_terms: D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000302:Adrenal Cortex / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D034284:Dynamin I / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D000080865:Formins; D006801:Humans; D016547:Kinesins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D055550:Protein Stability; D011987:Receptors, Steroid / Q000378:metabolism; D015398:Signal Transduction; D014404:Tubulin / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D000098825:Oxysterol Binding Proteins
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C108962:DIAPH1 protein, human; D000080865:Formins; C461064:OSBPL2 protein, human; D011987:Receptors, Steroid; D014404:Tubulin; D000098825:Oxysterol Binding Proteins; D000242:Cyclic AMP; D048049:Extracellular Signal-Regulated MAP Kinases; D034284:Dynamin I; D016547:Kinesins; D020742:rhoA GTP-Binding Protein