Limits...
Mitotic phosphorylation activates hepatoma-derived growth factor as a mitogen.

Everett AD, Yang J, Rahman M, Dulloor P, Brautigan DL - BMC Cell Biol. (2011)

Bottom Line: We found that HDGF in primary mouse aortic vascular smooth muscle cells (VSMC) was phosphorylated.Wild type HDGF was phosphorylated in asynchronous cells and substitution of S103, S165 and S202 to alanine each demonstrated a decrease in HDGF phosphorylation.We speculate that cell cycle regulated phosphorylation of HDGF may play an important role in vascular cell proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Cardiology Division, Johns Hopkins University, 600 N. Wolfe Street, Baltimore, MD 21287, USA. aeveret3@jhmi.edu

ABSTRACT

Background: Hepatoma-derived growth factor (HDGF) is a nuclear protein that is a mitogen for a wide variety of cells. Mass spectrometry based methods have identified HDGF as a phosphoprotein without validation or a functional consequence of this post-translational modification.

Results: We found that HDGF in primary mouse aortic vascular smooth muscle cells (VSMC) was phosphorylated. Wild type HDGF was phosphorylated in asynchronous cells and substitution of S103, S165 and S202 to alanine each demonstrated a decrease in HDGF phosphorylation. A phospho-S103 HDGF specific antibody was developed and demonstrated mitosis-specific phosphorylation. HDGF-S103A was not mitogenic and FACS analysis demonstrated a G2/M arrest in HDGF-S103A expressing cells, whereas cells expressing HDGF-S103D showed cell cycle progression. Nocodazole arrest increased S103 phosphorylation from 1.6% to 29% (P = 0.037).

Conclusions: Thus, HDGF is a phosphoprotein and phosphorylation of S103 is mitosis related and required for its function as a mitogen. We speculate that cell cycle regulated phosphorylation of HDGF may play an important role in vascular cell proliferation.

Show MeSH

Related in: MedlinePlus

HDGF is a phosphoprotein. Autoradiogram of HDGF immunoprecipitated from COS-7 cells that transiently expressed wild type rat GFP-HDGF (WT-HDGF) or S103A, S165A, or S202A-HDGF substitution mutations after in vivo [32P] orthophosphate labeling for two hours, and resolution by SDS-PAGE.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3094319&req=5

Figure 1: HDGF is a phosphoprotein. Autoradiogram of HDGF immunoprecipitated from COS-7 cells that transiently expressed wild type rat GFP-HDGF (WT-HDGF) or S103A, S165A, or S202A-HDGF substitution mutations after in vivo [32P] orthophosphate labeling for two hours, and resolution by SDS-PAGE.

Mentions: Previously we found multiple forms of HDGF by 2D gels and suspected this was due to a post-translational modification such as phosphorylation that could change the pI of the protein. The NetPhosK 1.0 computer algorithm [19] using statistical ranking identified S103 as the most likely candidate site for phosphorylation (Score 0.86). In addition, S165 and S202 were recently identified as phosphorylation sites in HDGF by mass spectrometry of HeLa nuclear and HT-29 cell extracts [16,17]. Sequence comparisons confirmed that S103, S165 and S202 are conserved in mouse, rat and human HDGF. COS-7 cells were transfected to express GFP fusions of wild type HDGF or S103A, S165A or S202A substitution HDGF polypeptides and metabolically radiolabeled with [32P] orthophosphate for 2 hours. The tagged proteins were recovered by anti-GFP immunoprecipitation and as shown in Figure 1, HDGF wt was phosphorylated and phosphorylation of the S103A, S165A and S202A were reduced relative to wild type (Figure 1) demonstrating that all three of these serines were kinase substrates.


Mitotic phosphorylation activates hepatoma-derived growth factor as a mitogen.

Everett AD, Yang J, Rahman M, Dulloor P, Brautigan DL - BMC Cell Biol. (2011)

HDGF is a phosphoprotein. Autoradiogram of HDGF immunoprecipitated from COS-7 cells that transiently expressed wild type rat GFP-HDGF (WT-HDGF) or S103A, S165A, or S202A-HDGF substitution mutations after in vivo [32P] orthophosphate labeling for two hours, and resolution by SDS-PAGE.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094319&req=5

Figure 1: HDGF is a phosphoprotein. Autoradiogram of HDGF immunoprecipitated from COS-7 cells that transiently expressed wild type rat GFP-HDGF (WT-HDGF) or S103A, S165A, or S202A-HDGF substitution mutations after in vivo [32P] orthophosphate labeling for two hours, and resolution by SDS-PAGE.
Mentions: Previously we found multiple forms of HDGF by 2D gels and suspected this was due to a post-translational modification such as phosphorylation that could change the pI of the protein. The NetPhosK 1.0 computer algorithm [19] using statistical ranking identified S103 as the most likely candidate site for phosphorylation (Score 0.86). In addition, S165 and S202 were recently identified as phosphorylation sites in HDGF by mass spectrometry of HeLa nuclear and HT-29 cell extracts [16,17]. Sequence comparisons confirmed that S103, S165 and S202 are conserved in mouse, rat and human HDGF. COS-7 cells were transfected to express GFP fusions of wild type HDGF or S103A, S165A or S202A substitution HDGF polypeptides and metabolically radiolabeled with [32P] orthophosphate for 2 hours. The tagged proteins were recovered by anti-GFP immunoprecipitation and as shown in Figure 1, HDGF wt was phosphorylated and phosphorylation of the S103A, S165A and S202A were reduced relative to wild type (Figure 1) demonstrating that all three of these serines were kinase substrates.

Bottom Line: We found that HDGF in primary mouse aortic vascular smooth muscle cells (VSMC) was phosphorylated.Wild type HDGF was phosphorylated in asynchronous cells and substitution of S103, S165 and S202 to alanine each demonstrated a decrease in HDGF phosphorylation.We speculate that cell cycle regulated phosphorylation of HDGF may play an important role in vascular cell proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Cardiology Division, Johns Hopkins University, 600 N. Wolfe Street, Baltimore, MD 21287, USA. aeveret3@jhmi.edu

ABSTRACT

Background: Hepatoma-derived growth factor (HDGF) is a nuclear protein that is a mitogen for a wide variety of cells. Mass spectrometry based methods have identified HDGF as a phosphoprotein without validation or a functional consequence of this post-translational modification.

Results: We found that HDGF in primary mouse aortic vascular smooth muscle cells (VSMC) was phosphorylated. Wild type HDGF was phosphorylated in asynchronous cells and substitution of S103, S165 and S202 to alanine each demonstrated a decrease in HDGF phosphorylation. A phospho-S103 HDGF specific antibody was developed and demonstrated mitosis-specific phosphorylation. HDGF-S103A was not mitogenic and FACS analysis demonstrated a G2/M arrest in HDGF-S103A expressing cells, whereas cells expressing HDGF-S103D showed cell cycle progression. Nocodazole arrest increased S103 phosphorylation from 1.6% to 29% (P = 0.037).

Conclusions: Thus, HDGF is a phosphoprotein and phosphorylation of S103 is mitosis related and required for its function as a mitogen. We speculate that cell cycle regulated phosphorylation of HDGF may play an important role in vascular cell proliferation.

Show MeSH
Related in: MedlinePlus