Regulation of progesterone receptor activity by cyclin dependent kinases 1 and 2 occurs in part by phosphorylation of the SRC-1 carboxyl-terminus

We described previously a novel role for cyclin A2/Cdk2 as a progesterone receptor (PR) coactivator. In reporter gene assays, cyclin A2 overexpression enhanced PR activity while inhibition of Cdk2 activity using the chemical inhibitor roscovitine or Cdk2 siRNA strongly inhibited PR activity. We demonstrate here that both Cdk1 and Cdk2 contribute to maximal induction of endogenous progestin responsive genes in T47D breast cancer cells. Our earlier studies suggested that the mechanism by which cyclin A2/Cdk2 enhances PR activity is via phosphorylation of steroid receptor coactivator-1 (SRC-1), which increases PR-SRC-1 interactions. To assess the importance of SRC-1 phosphorylation in the regulation of PR activity, SRC-1 was phosphorylated by cyclin A2/Cdk2 in vitro and seventeen phosphorylation sites were identified using biochemical techniques. We show that one of these sites, T1426 (adjacent to the C-terminal LXXLL nuclear receptor interaction motif), is an in vivo target of Cdks in mammalian cells and an in vitro target of Cdk1 and Cdk2. Phosphorylation of T1426 also contributes to SRC-1 coactivation potential, as mutation of the threonine target site to alanine results in reduced stimulation of PR activity by SRC-1. Together, these results suggest a role for Cdk1 and Cdk2 in the regulation of endogenous PR activity in part through phosphorylation of SRC-1.     

8-Bromo-cyclic AMP induces phosphorylation of two sites in SRC-1 that facilitate ligand-independent activation of the chicken progesterone receptor and are critical for functional cooperation between SRC-1 and CREB binding protein

Elevation of intracellular 8-bromo-cyclic AMP (cAMP) can activate certain steroid receptors and enhance the ligand-dependent activation of most receptors. During ligand-independent activation of the chicken progesterone receptor (cPR(A)) with the protein kinase A (PKA) activator, 8-bromo-cAMP, we found no alteration in cPR(A) phosphorylation (W. Bai, B. G. Rowan, V. E. Allgood, B. W. O'Malley, and N. L. Weigel, J. Biol. Chem. 272:10457-10463, 1997). To determine if other receptor-associated cofactors were targets of cAMP-dependent signaling pathways, we examined the phosphorylation of steroid receptor coactivator 1 (SRC-1). We detected a 1.8-fold increase in SRC-1 phosphorylation in transfected COS-1 cells incubated with 8-bromo-cAMP. Phosphorylation was increased on two mitogen-activated protein kinase (MAPK) sites, threonine 1179 and serine 1185. PKA did not phosphorylate these sites in vitro. However, blockage of PKA activity in COS-1 cells with the PKA inhibitor (PKI) prevented the 8-bromo-cAMP-mediated phosphorylation of these sites. Incubation of COS-1 cells with 8-bromo-cAMP resulted in activation of the MAPK pathway, as determined by Western blotting with antibodies to the phosphorylated (active) form of Erk-1/2, suggesting an indirect pathway to SRC-1 phosphorylation. Mutation of threonine 1179 and serine 1185 to alanine in COS-1 cells coexpressing cPR(A) and the GRE(2)E1bCAT reporter resulted in up to a 50% decrease in coactivation during both ligand-independent activation and ligand-dependent activation. This was due, in part, to loss of functional cooperation between SRC-1 and CREB binding protein for coactivation of cPR(A). This is the first demonstration of cross talk between a signaling pathway and specific phosphorylation sites in a nuclear receptor coactivator that can regulate steroid receptor activation.     

Endoplasmic reticulum-associated degradation mediated by MoHrd1 and MoDer1 is pivotal for appressorium development and pathogenicity of Magnaporthe oryzae

Most secretory proteins are folded and modified in the endoplasmic reticulum (ER); however, protein folding is error-prone, resulting in toxic protein aggregation and cause ER stress. Irreversibly misfolded proteins are subjected to ER-associated degradation (ERAD), modified by ubiquitination, and degraded by the 26S proteasome. The yeast ERAD ubiquitin ligase Hrd1p and multispanning membrane protein Der1p are involved in ubiquitination and transportation of the folding-defective proteins. Here, we performed functional characterization of MoHrd1 and MoDer1 and revealed that both of them are localized to the ER and are pivotal for ERAD substrate degradation and the ER stress response. MoHrd1 and MoDer1 are involved in hyphal growth, asexual reproduction, infection-related morphogenesis, protein secretion and pathogenicity of M. oryzae. Importantly, MoHrd1 and MoDer1 mediated conidial autophagic cell death and subsequent septin ring assembly at the appressorium pore, leading to abnormal appressorium development and loss of pathogenicity. In addition, deletion of MoHrd1 and MoDer1 activated the basal unfolded protein response (UPR) and autophagy, suggesting that crosstalk between ERAD and two other closely related mechanisms in ER quality control system (UPR and autophagy) governs the ER stress response. Our study indicates the importance of ERAD function in fungal development and pathogenesis of M. oryzae.