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Tyrosine-specific MAPK phosphatases and the control of ERK signaling in PC12 cells.

Noordman YE, Jansen PA, Hendriks WJ - J Mol Signal (2006)

Bottom Line: Spatio-temporal control of extracellular signal-regulated kinase (ERK) activity, a critical determinant of the cell's response to growth factors, requires timely dephosphorylation of its regulatory tyrosine and/or threonine residue by MAPK phosphatases.We found a single KIM-containing PTP to be endogenously expressed in rat PC12 cells: the transmembrane PTPRR isoform termed PCPTP1.Ectopic expression of cytosolic PTPRR isoforms, however, resulted in reduced EGF-induced ERK1/2 activity, an effect that was dependent on the phosphatase activity and the KIM-domain of these PTPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands. y.noordman@ncmls.ru.nl

ABSTRACT

Background: Spatio-temporal control of extracellular signal-regulated kinase (ERK) activity, a critical determinant of the cell's response to growth factors, requires timely dephosphorylation of its regulatory tyrosine and/or threonine residue by MAPK phosphatases. We studied the physiological role of kinase interaction motif (KIM)-containing protein tyrosine phosphatases (PTPs) in the control of EGF- and NGF-induced ERK activity in neuroendocrine PC12 cells.

Results: We found a single KIM-containing PTP to be endogenously expressed in rat PC12 cells: the transmembrane PTPRR isoform termed PCPTP1. Protein knock-down of PCPTP1, or fourfold overexpression of its mouse orthologue, PTPBR7, left EGF- and NGF-induced ERK1/2 activity in PC12 cells unaltered. Ectopic expression of cytosolic PTPRR isoforms, however, resulted in reduced EGF-induced ERK1/2 activity, an effect that was dependent on the phosphatase activity and the KIM-domain of these PTPs.

Conclusion: The finding that robust changes in tyrosine-specific MAPK phosphatase expression levels have minor effects on temporal ERK1/2 activity control in PC12 cells suggests that dual-specificity MAPK phosphatases may act as major regulators of growth factor-induced ERK1/2 signaling in these cells.

No MeSH data available.


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Assessment of PTPRR KIM domain phosphorylation. A) Lysates of PTPPBSγ expressing cells after overnight serum starvation (t = 0) and subsequent treatment with EGF, NGF or forskolin were subjected to immunoprecipitation with monoclonal antibody 6A6, and captured proteins were analyzed on blot using a phospho-PKA substrate antibody (upper panel) and 6A6 (lower panel). It was ascertained that the phospho-PKA substrate antibody remained unreactive towards PTPPBSγ mutant S94A (data not shown). B) Quantitative representation of phospho-PTPPBSγ levels determined as in A. Results, expressed as the ratio of the signals obtained with the phospho-PKA substrate antibody and the PTPRR antibody, are presented as mean values ± SEM (n = 3, Student's t-test, *p < 0.02). C) Representative image of PTPBR7 proteins, immunoprecipitated with antibody 6A6 from PTPBR7 expressing cells that received the indicated stimuli, as detected with phospho-PKA substrate antibody (upper panel) or 6A6 (lower panel). D) Quantitative representation of phospho-PTPBR7 levels as determined in C. Results are presented as mean values ± SEM (n = 3).
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Figure 5: Assessment of PTPRR KIM domain phosphorylation. A) Lysates of PTPPBSγ expressing cells after overnight serum starvation (t = 0) and subsequent treatment with EGF, NGF or forskolin were subjected to immunoprecipitation with monoclonal antibody 6A6, and captured proteins were analyzed on blot using a phospho-PKA substrate antibody (upper panel) and 6A6 (lower panel). It was ascertained that the phospho-PKA substrate antibody remained unreactive towards PTPPBSγ mutant S94A (data not shown). B) Quantitative representation of phospho-PTPPBSγ levels determined as in A. Results, expressed as the ratio of the signals obtained with the phospho-PKA substrate antibody and the PTPRR antibody, are presented as mean values ± SEM (n = 3, Student's t-test, *p < 0.02). C) Representative image of PTPBR7 proteins, immunoprecipitated with antibody 6A6 from PTPBR7 expressing cells that received the indicated stimuli, as detected with phospho-PKA substrate antibody (upper panel) or 6A6 (lower panel). D) Quantitative representation of phospho-PTPBR7 levels as determined in C. Results are presented as mean values ± SEM (n = 3).

Mentions: The binding of KIM-domain-containing phosphatases to their MAPK targets can be abrogated by the cAMP-dependent protein kinase PKA, through phosphorylation of a specific serine residue within the KIM-domain [9,10,12]. Intriguingly, current models on transient versus sustained ERK activation in PC12 cells incorporate an NGF-induced PKA-mediated signaling pathway [22]. To investigate whether this mechanism kept PTPRR isoforms from regulating ERK phosphorylation levels in PC12 cells, we investigated the phosphorylation status of the KIM domain serine residue in immunoprecipitated PTPRR proteins using an antibody directed against phosphorylated PKA substrates. Administration of the PKA activator forskolin was used to determine maximum levels of KIM-domain phosphorylation [12]. Phosphorylation levels of endogenous PCPTP1 in PC12 cells were, unfortunately, below detection level (data not shown). In PTPPBSγ-expressing PC12 cells about half of this phosphatase appeared phosphorylated following overnight serum starvation, and EGF or NGF administration even led to a significant decrease in the phosphorylation of the regulatory serine (Fig. 5A,B). In PTPBR7 expressing cells, however, the PTPBR7 KIM-domain seems maximally phosphorylated under all conditions tested (Fig. 5C, D), which may explain the negative findings regarding ERK1/2 activity modulation by this receptor-type PTPRR isoform.


Tyrosine-specific MAPK phosphatases and the control of ERK signaling in PC12 cells.

Noordman YE, Jansen PA, Hendriks WJ - J Mol Signal (2006)

Assessment of PTPRR KIM domain phosphorylation. A) Lysates of PTPPBSγ expressing cells after overnight serum starvation (t = 0) and subsequent treatment with EGF, NGF or forskolin were subjected to immunoprecipitation with monoclonal antibody 6A6, and captured proteins were analyzed on blot using a phospho-PKA substrate antibody (upper panel) and 6A6 (lower panel). It was ascertained that the phospho-PKA substrate antibody remained unreactive towards PTPPBSγ mutant S94A (data not shown). B) Quantitative representation of phospho-PTPPBSγ levels determined as in A. Results, expressed as the ratio of the signals obtained with the phospho-PKA substrate antibody and the PTPRR antibody, are presented as mean values ± SEM (n = 3, Student's t-test, *p < 0.02). C) Representative image of PTPBR7 proteins, immunoprecipitated with antibody 6A6 from PTPBR7 expressing cells that received the indicated stimuli, as detected with phospho-PKA substrate antibody (upper panel) or 6A6 (lower panel). D) Quantitative representation of phospho-PTPBR7 levels as determined in C. Results are presented as mean values ± SEM (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Assessment of PTPRR KIM domain phosphorylation. A) Lysates of PTPPBSγ expressing cells after overnight serum starvation (t = 0) and subsequent treatment with EGF, NGF or forskolin were subjected to immunoprecipitation with monoclonal antibody 6A6, and captured proteins were analyzed on blot using a phospho-PKA substrate antibody (upper panel) and 6A6 (lower panel). It was ascertained that the phospho-PKA substrate antibody remained unreactive towards PTPPBSγ mutant S94A (data not shown). B) Quantitative representation of phospho-PTPPBSγ levels determined as in A. Results, expressed as the ratio of the signals obtained with the phospho-PKA substrate antibody and the PTPRR antibody, are presented as mean values ± SEM (n = 3, Student's t-test, *p < 0.02). C) Representative image of PTPBR7 proteins, immunoprecipitated with antibody 6A6 from PTPBR7 expressing cells that received the indicated stimuli, as detected with phospho-PKA substrate antibody (upper panel) or 6A6 (lower panel). D) Quantitative representation of phospho-PTPBR7 levels as determined in C. Results are presented as mean values ± SEM (n = 3).
Mentions: The binding of KIM-domain-containing phosphatases to their MAPK targets can be abrogated by the cAMP-dependent protein kinase PKA, through phosphorylation of a specific serine residue within the KIM-domain [9,10,12]. Intriguingly, current models on transient versus sustained ERK activation in PC12 cells incorporate an NGF-induced PKA-mediated signaling pathway [22]. To investigate whether this mechanism kept PTPRR isoforms from regulating ERK phosphorylation levels in PC12 cells, we investigated the phosphorylation status of the KIM domain serine residue in immunoprecipitated PTPRR proteins using an antibody directed against phosphorylated PKA substrates. Administration of the PKA activator forskolin was used to determine maximum levels of KIM-domain phosphorylation [12]. Phosphorylation levels of endogenous PCPTP1 in PC12 cells were, unfortunately, below detection level (data not shown). In PTPPBSγ-expressing PC12 cells about half of this phosphatase appeared phosphorylated following overnight serum starvation, and EGF or NGF administration even led to a significant decrease in the phosphorylation of the regulatory serine (Fig. 5A,B). In PTPBR7 expressing cells, however, the PTPBR7 KIM-domain seems maximally phosphorylated under all conditions tested (Fig. 5C, D), which may explain the negative findings regarding ERK1/2 activity modulation by this receptor-type PTPRR isoform.

Bottom Line: Spatio-temporal control of extracellular signal-regulated kinase (ERK) activity, a critical determinant of the cell's response to growth factors, requires timely dephosphorylation of its regulatory tyrosine and/or threonine residue by MAPK phosphatases.We found a single KIM-containing PTP to be endogenously expressed in rat PC12 cells: the transmembrane PTPRR isoform termed PCPTP1.Ectopic expression of cytosolic PTPRR isoforms, however, resulted in reduced EGF-induced ERK1/2 activity, an effect that was dependent on the phosphatase activity and the KIM-domain of these PTPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands. y.noordman@ncmls.ru.nl

ABSTRACT

Background: Spatio-temporal control of extracellular signal-regulated kinase (ERK) activity, a critical determinant of the cell's response to growth factors, requires timely dephosphorylation of its regulatory tyrosine and/or threonine residue by MAPK phosphatases. We studied the physiological role of kinase interaction motif (KIM)-containing protein tyrosine phosphatases (PTPs) in the control of EGF- and NGF-induced ERK activity in neuroendocrine PC12 cells.

Results: We found a single KIM-containing PTP to be endogenously expressed in rat PC12 cells: the transmembrane PTPRR isoform termed PCPTP1. Protein knock-down of PCPTP1, or fourfold overexpression of its mouse orthologue, PTPBR7, left EGF- and NGF-induced ERK1/2 activity in PC12 cells unaltered. Ectopic expression of cytosolic PTPRR isoforms, however, resulted in reduced EGF-induced ERK1/2 activity, an effect that was dependent on the phosphatase activity and the KIM-domain of these PTPs.

Conclusion: The finding that robust changes in tyrosine-specific MAPK phosphatase expression levels have minor effects on temporal ERK1/2 activity control in PC12 cells suggests that dual-specificity MAPK phosphatases may act as major regulators of growth factor-induced ERK1/2 signaling in these cells.

No MeSH data available.


Related in: MedlinePlus