Identification and functional analysis of novel phosphorylation sites in Cx43 in rat primary granulosa cells

Abstract

The gap junctional intercellular communication mediated by Cx43 plays indispensable roles in both germ line development and postnatal folliculogenesis. In this study, we focused on the e¡ect of follicle-stimulating hormone (FSH) on the Cx43 protein in rat primary granulosa cells and found that FSH stimulation elevated the phosphorylation in addition to the protein level of Cx43. Serine residues in the carboxyl-terminal region were exclusively phosphorylated in this system and we identi¢ed Ser365, Ser368, Ser369 and Ser373 as major phosphorylation sites by FSH stimulation. A Cx43 variant containing mutations at all these serine residues was found to severely reduce dye transfer activity when assayed in HeLa cells. The present study revealed a novel regulatory mechanism of Cx43-mediated gap junctional intercellular communication through phosphorylation in the carboxyl-terminus.

PKA Implicated in the Phosphorylation of Cx43 Induced by Stimulation with FSH in Rat Granulosa Cells

“…We previously found that FSH accelerated trafficking of Cx43 and accumulation of this protein i n g a p j u n c t i o n a l p l a q u e s a c c o m p a n y i n g phosphorylation in rat granulosa cells [40]. We further identified Ser365, Ser368, Ser369, and Ser373 as major sites of phosphorylation, and revealed the significance of serine phosphorylation in Cx43-mediated channel activity [16]. Namely, when all four serine residues were substituted with alanine, Cx43 appeared to lose dye transfer activity.…”

“…We previously found that recombinant Cx43-CT could be phosphorylated in vitro using rat granulosa cell lysate, and the sites of phosphorylation were identical to those observed in vivo [16]. We, therefore, expressed and purified 6x Histidinetagged Cx43-CT (Fig.…”

“…In fact, we found that FSH promoted phosphorylation of Cx43 in rat primary granulosa cells [16]. We further identified Ser365, Ser368, Ser369, and Ser373 in the carboxyterminal tail as major sites of phosphorylation on s t i m u l a t i o n w i t h F S H , a n d f o u n d t h a t phosphorylation of these four residues was essential for channel activity [16].…”

“…1) were expressed in E. coli. and purified as described previously [16]. Cell lysates were prepared as follows.…”

The effects of connexin phosphorylation on gap junctional communication

“…serines 365, 368, 369 and 373) in response to folliclestimulating hormone (FSH), probably at least partially through a PKA mechanism ( Fig. 1 and Table 1) (Yogo, Ogawa, Akiyama, Ishida, & Takeya, 2002). As shown in Table 1, there is experimental evidence that Cx43 can be phosphorylated on at least 12 of the 21 serines and two of the tyrosines of the cytoplasmic tail region (amino acids 250-382).…”