d155a cdna  (Addgene inc)

Journal: Molecular and Cellular Biology

Article Title: Pin1 Facilitates the Phosphorylation-Dependent Ubiquitination of SF-1 To Regulate Gonadotropin β-Subunit Gene Transcription

doi: 10.1128/mcb.00807-09

Figure Lengend Snippet: FIG. 6. GnRH treatment stimulates SF-1 ubiquitination. (A) LT2 cell lysates after transfection with FLAG–SF-1 and exposure of some cells to 100 nM GnRH for 6 h were precipitated with anti-FLAG M2 beads. The IP samples were analyzed using anti-FLAG, Rb anti-FLAG, and anti-Ub Ab. (B) Western blotting detected phosphorylated ERK (pERK) in LT2 cells after exposure to 100 nM GnRH for 0 to 4 h and/or 1 M U0126; total ERK is shown as a loading control. (C and D) LT2 cell lysates after transfection with WT FLAG–SF-1 or SF-1 S203A, some of which were exposed to 100 nM GnRH and 1 M U0126 (C) or 15 M roscovitine (ROS) (D) for 0 or 4 h, were precipitated with anti-FLAG M2 beads. The input and IP samples were analyzed using anti-FLAG, anti-Ub, and anti-GAPDH Ab. (E) LT2 cell lysates after transfection with FLAG–SF-1 together with pcDNA3, pcDNA3 CDK7 HA, or pcDNA3 CDK7 D155A HA were precipitated with anti-FLAG M2 beads. The input and IP samples were analyzed by Western blotting. Monoubiquitinated SF-1 is marked with arrows on the left.

Article Snippet: Pitx1, SF-1, Egr-1, ubiquitin, and SUMO1 sequences were inserted into pxj40-HA, pxj40-FLAG, and pxj40-Myc (gifts from B. C. Low, National University of Singapore). pxj40-GFP was used to construct pxj40 GFP SF-1 and pxj40 GFP SF-1 K119R. pcDNA3 CDK7 HA (P#633) and pcDNA3 CDK7 D155A HA (P#812) were purchased from Addgene.

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Control

Journal: Molecular and Cellular Biology

Article Title: Pin1 Facilitates the Phosphorylation-Dependent Ubiquitination of SF-1 To Regulate Gonadotropin β-Subunit Gene Transcription

doi: 10.1128/mcb.00807-09

Figure Lengend Snippet: FIG. 10. Model of regulation of gonadotropin -subunit gene transcription by Pin1. GnRH activates PKC and PKA, either of which might phosphorylate Pin1 at Ser 16, causing its nuclear export. However, GnRH also elevates intracellular calcium levels and activates calcineurin, which dephosphorylates Pin1, allowing it to translocate back into the nucleus. Inside the nucleus Pin1, interacts with its target transcription factors, including SF-1, Pitx1, and Egr-1, enhancing their transcriptional activity toward the gonadotropin -subunit genes. DeSUMOylation of SF-1 at K194, which occurs through an unknown mechanism, results in enhanced phosphorylation of SF-1 at Ser203 by CDK7 (67). DeSUMOylation at K119 and GnRH-activated ERK1/2 also increase SF-1 phosphorylation. Phosphorylation of SF-1 and its subsequent isomerization by Pin1 promote ubiquitination of SF-1 at K119, facilitating the interaction between SF-1 and Pitx1 (and possibly other cofactors), to upregulate gonadotropin -subunit gene transcription. The proteasomal degradation of ubiquitinated SF-1 in the cytosol is a putative event, for which we do not yet have direct proof. Dashed lines represent pathways that have or may have (for PKA and PKC phosphorylation of Pin1) intermediate elements that are not shown.

Article Snippet: Pitx1, SF-1, Egr-1, ubiquitin, and SUMO1 sequences were inserted into pxj40-HA, pxj40-FLAG, and pxj40-Myc (gifts from B. C. Low, National University of Singapore). pxj40-GFP was used to construct pxj40 GFP SF-1 and pxj40 GFP SF-1 K119R. pcDNA3 CDK7 HA (P#633) and pcDNA3 CDK7 D155A HA (P#812) were purchased from Addgene.

Techniques: Activity Assay, Phospho-proteomics, Ubiquitin Proteomics