Dephosphorylation of MAP2D enhances its binding to vimentin in preovulatory ovarian granulosa cells

Abstract

Preovulatory granulosa cells express microtubule-associated protein(MAP) 2D. Luteinizing hormone(LH)/choriogonadotropin(CG) receptor activation by human(h) CG promotes dephosphorylation of MAP2D on Thr256/Thr259. We sought to evaluate the association of MAP2D with the cytoskeleton and the effect of hCG on this association. MAP2D partially co-localizes by confocal immunofluorescence microscopy with the vimentin intermediate filament and microtubule cytoskeletons in naive cells. In vitro binding studies show that MAP2D binds directly to vimentin and β-tubulin. Phosphorylation of recombinant MAP2D on Thr256/Thr259 that mimics the phosphorylation status of MAP2D in naive cells reduces binding of MAP2D to vimentin and tubulin 2- and 3-fold, respectively. hCG promotes PKA-dependent phosphorylation of vimentin(Ser32/Ser38), increased binding of vimentin to MAP2D, and contraction of granulosa cells with reorganization of vimentin filaments and MAP2D from the periphery into a thickened layer surrounding the nucleus and into prominent cellular extensions. Chemical disruption of vimentin filament organization increased progesterone production. Together these results suggest that hCG-stimulated dephosphorylation of MAP2D at Thr256/Thr259, phosphorylation of vimentin at Ser38/Ser72, and resulting enhanced binding of MAP2D to vimentin may contribute to the progesterone synthetic response required for ovulation.