cAMP-stimulated phosphorylation of diaphanous 1 regulates protein stability and interaction with binding partners in...

Area of Science:

• Cellular dynamics
• Molecular biology
• Biochemistry

Background:

• Diaphanous homologue 1 (DIAPH1) is a Rho effector protein regulating cellular dynamics via microfilament and microtubule interactions.
• Previous studies linked DIAPH1 to cortisol production by controlling mitochondrial movement, influenced by ACTH/cAMP signaling and RhoA interaction.
• Understanding DIAPH1's regulatory mechanisms is key to cellular signaling pathways.

Purpose of the Study:

• To identify DIAPH1 binding partners using mass spectrometry.
• To investigate the role of DIAPH1 phosphorylation at Thr-759 in its stability and function.
• To elucidate the impact of DIAPH1 phosphorylation on mitochondrial trafficking.

Main Methods:

• Mass spectrometry was employed to identify DIAPH1 interacting proteins.
• Site-directed mutagenesis (T759A substitution) was used to assess the role of Thr-759 phosphorylation.
• Cellular assays measured mitochondrial movement rates in response to signaling pathway activation and DIAPH1 mutants.

Main Results:

• DIAPH1 was found to interact with RhoA, dynamin-1, kinesin, β-tubulin, β-actin, ORP2, and ORP10.
• DIAPH1 is phosphorylated at Thr-759 by ERK in response to Bt2cAMP stimulation.
• The T759A mutation stabilized DIAPH1, reduced interactions with kinesin, ORP2, and actin, and impaired mitochondrial movement.

Conclusions:

• Phosphorylation of DIAPH1 at Thr-759 is a critical regulatory mechanism for its stability and function.
• This phosphorylation event modulates DIAPH1's interactions with specific binding partners, affecting mitochondrial dynamics.
• The findings highlight a novel regulatory pathway involving ERK, DIAPH1 phosphorylation, and cellular signaling.