Phosphatase-mediated crosstalk control of ERK and p38 MAPK signaling in corneal epithelial cells.
OBJECTIVE
To test the hypothesis that the protein phosphatases PP2A and MKP-1 are involved in controlling epidermal growth factor (EGF)-induced increases in rabbit corneal epithelial cell (RCEC) migration by mediating crosstalk between signaling pathways eliciting EGF receptor control of migration and proliferation.


METHODS
Western blot analysis was used to determine the phosphorylation status of Erk1/2, p38, and the mitogen-activated protein kinase (MAPK) kinase (MEK1/2) using inhibitors of Erk1/2 or p38 and dominant-negative (d/n) Erk1 or d/n p38 cell lines. Coimmunoprecipitation was used to evaluate protein phosphatase (PP)2A and Erk1/2 interaction. Short-interfering RNA (siRNA) transfection was performed to analyze the involvement of MAPK phosphatase (MKP)-1 in crosstalk. Scratch-wound assay was used to determine EGF-dependent effects on cell migration.


RESULTS
EGF (10 ng/mL) induced changes in activation of Erk1/2 and p38, which were enhanced by inhibition with 10 microM SB203580 and 10 muM PD98059, respectively. PP inhibition with sodium orthovanadate (100 microM), okadaic acid (10 nM), or Ro 31-8220 (10 microM) resulted in larger and more prolonged increases in the phosphorylation status of Erk1/2 and p38. After 1 hour, EGF induced 14-fold increases in MKP-1 protein expression. After MKP-1 siRNA transfection, EGF had induced a similar pattern of changes in the phosphorylation status in Erk1/2 and p38 following PP inhibition. EGF-induced cell migration was enhanced by Erk1/2 pathway inhibition and was accentuated after PP inhibition. Conversely, p38 pathway inhibition eliminated this response.


CONCLUSIONS
EGF-induced changes in Erk1/2 and p38 phosphorylation status are dependent on PP-mediated crosstalk. This control modulates the magnitude of growth factor-induced increases in corneal epithelial cell migration.
Wang
Zheng
Z
Department of Biological Sciences, State University of New York, College of Optometry, New York, New York 10036, USA.
Yang
Hua
H
Tachado
Souvenir D
SD
Capó-Aponte
José E
JE
Bildin
Victor N
VN
Koziel
Henry
H
Reinach
Peter S
PS
D000818:Animals; D015153:Blotting, Western; D018929:Cell Culture Techniques; D018797:Cell Cycle Proteins / Q000502:physiology; D002465:Cell Movement; D054638:Dual Specificity Phosphatase 1; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D017874:Immediate-Early Proteins / Q000502:physiology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D017027:Protein Tyrosine Phosphatases / Q000502:physiology; D011817:Rabbits; D020239:Receptor Cross-Talk / Q000502:physiology*; D015398:Signal Transduction; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D018797:Cell Cycle Proteins; D017874:Immediate-Early Proteins; C086766:Ppp2r1b protein, mouse; D004815:Epidermal Growth Factor; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases