Related Experiment Videos
SHP-1 phosphatase C-terminus interacts with novel substrates p32/p30 during erythropoietin and interleukin-3
1Department of Cancer Biology, The Lerner Research Institute of the Cleveland Clinic Foundation, Cleveland, OH, USA.
Abstract:
SHP-1 protein tyrosine phosphatase is a critical negative regulator of mitogenic signaling, as demonstrated by the heightened growth responses to hematopoietic growth factors in hematopoietic cells of motheaten mice, which lack functional SHP-1 expression due to mutations in the SHP-1 gene. The mitogenic signaling molecules dephosphorylated by SHP-1 have not been fully identified. We detected two proteins (p32/p30) that are hyperphosphorylated in a DA3/erythropoietin receptor (EpoR) cell line that expresses a mutant containing the SHP-1 C-terminus that suppresses the function of the endogenous phosphatase and induces hyperproliferative responses to interleukin-3 (IL-3) and Epo. Hyperphosphorylated p32/p30 are also detected in motheaten hematopoietic cells, demonstrating an association of p32/p30 hyperphosphorylation with SHP-1-deficiency and growth factor-hyperresponsiveness. The hyperphosphorylated p32/30 associate with SHP-1 via its C-terminus, because they coimmunoprecipitate with the phosphatase and the C-terminal mutant and they bind in vitro to a synthetic peptide of the mutant but not the GST fusion proteins of SHP-1 SH2 domains. Induction of p32/p30 phosphorylation by IL-3 or Epo occurs mainly at 2 to 18 hours poststimulation in the DA3/EpoR cell line, indicating p32/p30 as novel signaling molecules during cell cycle progression. These data demonstrate a function for the SHP-1 C-terminus in recruiting potential substrates p32/p30 and suggest that SHP-1 may regulates mitogenic signaling by dephosphorylating p32/p30.
Insights
SHP-1 protein tyrosine phosphatase regulates cell growth by dephosphorylating novel signaling molecules p32/p30. SHP-1 deficiency leads to hyperphosphorylation of p32/p30 and hyper-responsiveness to growth factors.
Area of Science:
- Cellular signaling
- Protein tyrosine phosphatases
- Hematopoiesis
Background:
- SHP-1 protein tyrosine phosphatase (PTP) is a key negative regulator of mitogenic signaling pathways.
- SHP-1 deficiency, observed in motheaten mice, results in heightened cellular responses to hematopoietic growth factors.
- The specific substrates dephosphorylated by SHP-1 remain largely unidentified.
Purpose of the Study:
- To identify novel substrates of SHP-1 involved in mitogenic signaling.
- To investigate the role of the SHP-1 C-terminus in substrate recruitment and regulation.
- To understand the mechanism linking SHP-1 deficiency to hyperproliferative responses.
Main Methods:
- Utilized a DA3/erythropoietin receptor (EpoR) cell line expressing a dominant-negative SHP-1 C-terminal mutant.
- Detected hyperphosphorylated proteins p32/p30 in cell lines and motheaten hematopoietic cells.
- Performed co-immunoprecipitation and in vitro binding assays to study protein interactions.
Main Results:
- Identified two proteins, p32/p30, that are hyperphosphorylated in SHP-1-deficient cells and cell lines with a dominant-negative SHP-1 mutant.
- Demonstrated that hyperphosphorylated p32/p30 associate with the C-terminus of SHP-1.
- Showed that IL-3 and Epo induce p32/p30 phosphorylation, suggesting their role in cell cycle progression.
Conclusions:
- p32/p30 are novel signaling molecules whose phosphorylation is regulated by SHP-1.
- The C-terminus of SHP-1 plays a crucial role in recruiting p32/p30 as potential substrates.
- SHP-1 likely regulates mitogenic signaling by dephosphorylating p32/p30, thus controlling cell proliferation.