The epidermal growth factor receptor (EGFR) and proline rich tyrosine kinase 2 (PYK2) are involved in tissue factor dependent factor VIIa signalling in HaCaT cells

Thromb Haemost. 2004 Jul;92(1):13-22. doi: 10.1160/TH03-08-0549.

Abstract

Binding of the coagulation protease factor VIIa to its receptor Tissue Factor (TF) induces intracellular signals in several cell types including HaCaT keratinocytes. TF belongs to the cytokine receptor family, but is most likely not alone in transferring the complete TF/FVIIa signal over the plasma membrane. The protease activated receptor PAR2 is involved in factor VIIa and factor Xa signal transduction. Our results indicate that the epidermal growth factor receptor (EGFR) and the proline rich tyrosine kinase 2 (PYK2) participate in TF/FVIIa signalling as formation of the TF/FVIIa complex increased the phosphorylation of these proteins. Both FVIIa protease activity and available TF were necessary for generation of the signal. Increased tyrosine phosphorylation of the EGFR was observed following TF/FVIIa complex formation on the cell surface. The EGFR kinase inhibitor tyrphostin AG1478 abrogated the TF/FVIIa-complex induced MAP kinase activation and mRNA increase of egr-1, heparin-binding EGF, and interleukin-8 following FVIIa addition. Using specific antibodies, increased phosphorylation of PYK2 tyrosine residues 402 and 580 was observed. The first site is the major autophosphorylation site and the docking site for Src family kinases. The second site is important for the kinase activity. The Src family kinase Yes and the tyrosine phosphatase SHP-2 were detected in immunoprecipitates using either anti-PYK2 or anti-EGFR antibodies. Their coprecipitation with EGFR increased in the presence of FVIIa. Moreover, the coprecipitation of EGFR and PYK2 increased with FVIIa stimulation. Together, these data suggest that EGFR, PYK2, Yes, and SHP-2 are involved in transduction of the TF/FVIIa signal possibly via transactivation of the EGF receptor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Cell Line
  • DNA-Binding Proteins / genetics
  • Early Growth Response Protein 1
  • Epidermal Growth Factor / genetics
  • ErbB Receptors / metabolism*
  • Factor VIIa / metabolism*
  • Factor VIIa / pharmacology
  • Focal Adhesion Kinase 2
  • Heparin-binding EGF-like Growth Factor
  • Humans
  • Immediate-Early Proteins / genetics
  • Intercellular Signaling Peptides and Proteins
  • Interleukin-8 / genetics
  • Keratinocytes / drug effects
  • Keratinocytes / metabolism
  • Phosphorylation
  • Protein-Tyrosine Kinases / antagonists & inhibitors
  • Protein-Tyrosine Kinases / chemistry
  • Protein-Tyrosine Kinases / metabolism*
  • Quinazolines
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / pharmacology
  • Signal Transduction / drug effects
  • Thromboplastin / metabolism*
  • Transcription Factors / genetics
  • Tyrphostins / pharmacology

Substances

  • DNA-Binding Proteins
  • EGR1 protein, human
  • Early Growth Response Protein 1
  • HBEGF protein, human
  • Heparin-binding EGF-like Growth Factor
  • Immediate-Early Proteins
  • Intercellular Signaling Peptides and Proteins
  • Interleukin-8
  • Quinazolines
  • RNA, Messenger
  • Recombinant Proteins
  • Transcription Factors
  • Tyrphostins
  • RTKI cpd
  • Epidermal Growth Factor
  • Thromboplastin
  • ErbB Receptors
  • Protein-Tyrosine Kinases
  • Focal Adhesion Kinase 2
  • Factor VIIa