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

Abstract

Preovulatory granulosa cells express the low-molecular-mass MAP2D variant of microtubule-associated protein 2 (MAP2). Activation of the luteinizing hormone choriogonadotropin receptor by human choriogonadotropin (hCG) promotes dephosphorylation of MAP2D on Thr256 and Thr259. We sought to evaluate the association of MAP2D with the cytoskeleton, and the effect of hCG on this association. MAP2D partially colocalized, as assessed by confocal immunofluorescence microscopy, with the vimentin intermediate filament and microtubule cytoskeletons in naive cells. In vitro binding studies showed that MAP2D bound directly to vimentin and β-tubulin. Phosphorylation of recombinant MAP2D on Thr256 and Thr259, which mimics the phosphorylation status of MAP2D in naive cells, reduces binding of MAP2D to vimentin and tubulin by two-and three-fold, respectively. PKA-dependent phosphorylation of vimentin (Ser32 and Ser38) promoted binding of vimentin to MAP2D and increased 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. Taken together, these results suggest that hCG-stimulated dephosphorylation of MAP2D at Thr256 and Thr259, phosphorylation of vimentin at Ser38 and Ser72, and the resulting enhanced binding of MAP2D to vimentin might contribute to the progesterone synthetic response required for ovulation.

Phosphoproteomics Analysis Identifies Novel Candidate Substrates of the Nonreceptor Tyrosine Kinase, Src-related Kinase Lacking C-terminal Regulatory Tyrosine and N-terminal Myristoylation Sites (SRMS)

“…Vimentin was predominantly found to localize to cytoplasmic tubular networks (Fig. 9D), consistent with previous reports describing these as vimentin intermediate filaments (VIFs) (63,64). Importantly, we observed that GFP-vimentin and mCherry-wild type SRMS colocalized in the cytoplasm of HEK293 cells (Fig.…”

The Ovulatory Signal Precipitates LRH-1 Transcriptional Switching Mediated by Differential Chromatin Accessibility

“…Another important role of the cytoskeleton in the regulation of steroidogenesis has been reported (Sewer and Li, 2008). More particularly, it was shown that after the ovulatory stimulus in granulosa cells, a coordinated and precise regulation of vimentin, an important protein of the cytoskeleton, contributes to cholesterol transport and progesterone synthesis required for ovulation (Flynn et al, 2016;Shen et al, 2012). Together, these data are consistent with previous studies showing defective cumulus expansion and ovulation (Bertolin et al, 2014Duggavathi et al, 2008) and disrupted progesterone production (Zhang et al, 2013) that are the sequelae to depletion of LRH-1 in granulosa cells.…”

G protein-coupled receptors (GPCRs) That Signal via Protein Kinase A (PKA) Cross-talk at Insulin Receptor Substrate 1 (IRS1) to Activate the phosphatidylinositol 3-kinase (PI3K)/AKT Pathway

“…Granulosa, FRTL, and MCF7 Cell Culture-Preantral GCs were isolated from 24-day-old immature Sprague-Dawley rats primed with estrogen and cultured under serum-free conditions as described previously (31). PO GCs were isolated from 24-day-old immature rats injected with pregnant mare's serum gonadotropin 48 h prior to isolation and cultured under serumfree conditions (76). Rats were from a breeder colony (originally from Charles River Laboratories) maintained in accordance with the Animal Welfare Act by protocols approved by the Washington State University Animal Care and Use Committee.…”