Protein kinase Cdelta-mediated phosphorylation of phospholipase D controls integrin-mediated cell spreading

Mol Cell Biol. 2010 Nov;30(21):5086-98. doi: 10.1128/MCB.00443-10. Epub 2010 Aug 23.

Abstract

Integrin signaling plays critical roles in cell adhesion, spreading, and migration, and it is generally accepted that to regulate these integrin functions accurately, localized actin remodeling is required. However, the molecular mechanisms that control the targeting of actin regulation molecules to the proper sites are unknown. We previously demonstrated that integrin-mediated cell spreading and migration on fibronectin are dependent on the localized activation of phospholipase D (PLD). However, the mechanism underlying PLD activation by integrin is largely unknown. Here we demonstrate that protein kinase Cδ (PKCδ) is required for integrin-mediated PLD signaling. After integrin stimulation, PKCδ is activated and translocated to the edges of lamellipodia, where it colocalizes with PLD2. The abrogation of PKCδ activity inhibited integrin-induced PLD activation and cell spreading. Finally, we show that Thr566 of PLD2 is directly phosphorylated by PKCδ and that PLD2 mutation in this region prevents PLD2 activation, PLD2 translocation to the edge of lamellipodia, Rac translocation, and cell spreading after integrin activation. Together, these results suggest that PKCδ is a primary regulator of integrin-mediated PLD activation via the direct phosphorylation of PLD, which is essential for directing integrin-induced cell spreading.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Base Sequence
  • COS Cells
  • Cell Adhesion / physiology*
  • Cell Movement / physiology*
  • Chlorocebus aethiops
  • Guanosine Triphosphate / metabolism
  • Humans
  • In Vitro Techniques
  • Integrins / physiology*
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phospholipase D / antagonists & inhibitors
  • Phospholipase D / chemistry
  • Phospholipase D / genetics
  • Phospholipase D / metabolism*
  • Phosphorylation
  • Protein Kinase C-delta / antagonists & inhibitors
  • Protein Kinase C-delta / genetics
  • Protein Kinase C-delta / metabolism*
  • Pseudopodia / physiology
  • RNA, Small Interfering / genetics
  • Rats
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Threonine / chemistry
  • Transfection
  • rac1 GTP-Binding Protein / metabolism

Substances

  • Integrins
  • RAC1 protein, human
  • RNA, Small Interfering
  • Recombinant Fusion Proteins
  • Threonine
  • Guanosine Triphosphate
  • Protein Kinase C-delta
  • phospholipase D2
  • Phospholipase D
  • phospholipase D1
  • rac1 GTP-Binding Protein