Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 8

1.
Fig. 8.

Fig. 8. From: Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function.

Proposed model for the regulation of Pin1 by MLK3.

Velusamy Rangasamy, et al. Proc Natl Acad Sci U S A. 2012 May 22;109(21):8149-8154.
2.
Fig. 3.

Fig. 3. From: Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function.

Phosphorylated Pin1 interacts with MLK3. HEK293 cells were transfected with FLAG-tagged MLK3 along with GFP-tagged WT or different mutants of Pin1, as indicated. MLK3 (A) and Pin1 (B) were immunoprecipitated using anti-FLAG or anti-GFP antibodies, respectively, and blotted with anti-GFP for Pin1 and anti-FLAG for MLK3.

Velusamy Rangasamy, et al. Proc Natl Acad Sci U S A. 2012 May 22;109(21):8149-8154.
3.
Fig. 7.

Fig. 7. From: Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function.

Phosphorylated Pin1 is elevated in breast tumors and localizes in the nucleus. (A) Normalized Pin1 expression from six pairs of breast tumors (T) and matching normal (N) breast tissue extracts that were blotted, either with Pin1 pSer138 or MLK3 or with Pin1 antibodies. (B and C) Commercial tissue microarrays containing normal and cancer tissues (B) or normal, benign, and cancer tissues (C) were stained with Pin1 pSer138 antibodies and counted. (D) Representative normal and cancerous breast tissue stained with Pin1 pSer138 showing nuclear localization (arrowheads).

Velusamy Rangasamy, et al. Proc Natl Acad Sci U S A. 2012 May 22;109(21):8149-8154.
4.
Fig. 5.

Fig. 5. From: Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function.

The cell-cycle function of Pin1 is regulated via MLK3-mediated phosphorylation. (A) HeLa cells were transfected with either empty GFP vector or the indicated GFP-Pin1 expression vectors. The cells were synchronized using double-thymidine block and released into the cell cycle with 5% (vol/vol) FBS. The cell-cycle progression of GFP-positive HeLa cells was determined after PI staining by FACS analysis. (B) HeLa cells were transfected with the indicated Pin1 expression vectors, and cyclin B1-associated Cdc2 kinase activity was determined from cyclin B1 immunoprecipitates, using histone H1 as substrate. The WCEs were blotted with the indicated antibodies for the total expression of endogenous Cdc2, MLK3, and cyclin B1 and recombinant GFP-Pin1 proteins.

Velusamy Rangasamy, et al. Proc Natl Acad Sci U S A. 2012 May 22;109(21):8149-8154.
5.
Fig. 1.

Fig. 1. From: Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function.

MLK3 associates with Pin1. (A) Endogenous MLK3 from breast cancer cell lines was immunoprecipitated, and associated endogenous Pin1 was detected by anti-Pin1 antibody. The total expression of Pin1 and MLK3 was detected in whole-cell extracts (WCEs). IB, immunoblotting; IP, immunoprecipitation. (B) HEK293 cells were transfected with GFP-Pin1 (WT) along with either full-length or different deletion mutants of GST-MLK3, as indicated. The MLK3 proteins were pulled down with GSH beads and blotted with anti-GFP antibody to detect associated GFP-Pin1. The WCEs were blotted with anti-GFP for Pin1 and anti-GST for MLK3 expression. (C and D) HEK293 cells were transfected with FLAG-tagged MLK3 along with full-length (FL) or GFP-tagged different domains (i.e., WW and PPI) of Pin1. MLK3 (C) and Pin1 (D) were immunoprecipitated using anti-FLAG and anti-GFP antibodies, respectively, and blotted with anti-GFP for Pin1 and anti-FLAG for MLK3 detection.

Velusamy Rangasamy, et al. Proc Natl Acad Sci U S A. 2012 May 22;109(21):8149-8154.
6.
Fig. 4.

Fig. 4. From: Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function.

Catalytic activity and nuclear translocation of Pin1 is increased upon phosphorylation by MLK3. (A) Activity of recombinant WT, S138A, and S138E Pin1 mutants expressed in Pin1−/− MEFs was measured by taking 2 μg of whole-cell extracts. pNA, p-nitroanilide. (B) Activity of in vitro phosphorylated WT or S138A Pin1 proteins was measured upon phosphorylation by purified MLK3. Data shown are mean ± SEM, (n = 4). (C and D) The nuclear (N) and cytoplasmic (C) distributions of recombinant WT or Pin1 mutants in HeLa cells were determined, either expressed alone (C) or coexpressed with MLK3 (D). The nuclear and cytoplasmic fractions were also blotted with GAPDH and lamin A/C as cytoplasmic and nuclear markers. (E) The localization of GFP-tagged WT, S138A, and S138E Pin1 was visualized in HeLa cells by confocal microscopy.

Velusamy Rangasamy, et al. Proc Natl Acad Sci U S A. 2012 May 22;109(21):8149-8154.
7.
Fig. 2.

Fig. 2. From: Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function.

MLK3 directly phosphorylates Pin1. (A) In vitro phosphorylation of bacterially expressed Pin1 (5 μg) was performed using increasing concentrations of either recombinant WT or kinase-dead (K-A) MLK3, expressed in HEK293 cells. (B) Direct phosphorylation of bacterially expressed full-length or different domains of Pin1 was performed in the presence of purified recombinant GST-MLK3 (GST-rMLK3), produced in baculovirus. GST-Pin1 and GST-rMLK3 were blotted with anti-GST antibody. (C and D) Serine 138 on Pin1 was identified as an MLK3-specific phosphorylation site by mass spectroscopy and mutated to alanine to make phospho-deficient protein in bacteria. The purified Pin1 proteins were used in an in vitro phosphorylation assay with purified GST-rMLK3 protein (C), and these phosphorylated Pin1 proteins were analyzed by 2D peptide mapping (D). (E) Pin1 (WT) or S138A expression vectors were coexpressed either with WT or kinase-dead MLK3 in HEK293 cells. The recombinant GFP-Pin1 was blotted with Pin1 pSer138 antibody. The kinase activities are expressed as phosphorimager units (PI-Units).

Velusamy Rangasamy, et al. Proc Natl Acad Sci U S A. 2012 May 22;109(21):8149-8154.
8.
Fig. 6.

Fig. 6. From: Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function.

Cyclin D1 expression and centrosome duplication are regulated by phospho-mimetic Pin1. (A) Cyclin D1 protein stability was determined in the NIH 3T3 cell line, expressing indicated GFP-Pin1 expression vectors. The cells were treated with cyclohexamide and harvested at the times indicated. The total cell lysates were blotted with cyclin D1, β-actin, and GFP antibodies (for GFP-Pin1). (B) Cyclin D1 reporter activity was determined in HeLa cells, and the reporter was transfected with the indicated Pin1 plasmids along with cyclin D1 luciferase reporter vector. The activity of the reporter luciferase is expressed relative to vector-transfected cells. Data shown are mean ± SEM, (n = 3). (C) The centrosome duplication assay in NIH 3T3 cells, transfected with the indicated Pin1 or vector plasmids, was performed upon arresting them at G1/S phase. The centrosomes were stained with anti–γ-tubulin and analyzed by fluorescence microscopy.

Velusamy Rangasamy, et al. Proc Natl Acad Sci U S A. 2012 May 22;109(21):8149-8154.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk