Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility.
Protein kinase C (PKC) alpha has been implicated in beta1 integrin-mediated cell migration. Stable expression of PKCalpha is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein-PKCalpha and beta1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCalpha is shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy showed an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCalpha-induced cell migration. We provide the first evidence that PKCalpha or a PKCalpha-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase-ezrin molecular complex in vivo.
Ng
T
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Richard Dimbleby Department of Cancer Research, St Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, UK. T.Ng@icrf.icnet.uk
Parsons
M
M
Hughes
W E
WE
Monypenny
J
J
Zicha
D
D
Gautreau
A
A
Arpin
M
M
Gschmeissner
S
S
Verveer
P J
PJ
Bastiaens
P I
PI
Parker
P J
PJ
D019943:Amino Acid Substitution; D001943:Breast Neoplasms; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D002867:Chromones / Q000494:pharmacology; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D049452:Green Fluorescent Proteins; D006801:Humans; D019012:Integrin beta1 / Q000502:physiology*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D018613:Microscopy, Confocal; D009025:Morpholines / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry; D014407:Tumor Cells, Cultured; D014945:Wound Healing / Q000502:physiology*; D000099149:Ezrin
D002867:Chromones; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D049452:Green Fluorescent Proteins; D019012:Integrin beta1; D007527:Isoenzymes; D008164:Luminescent Proteins; D009025:Morpholines; D015240:Phorbol 12,13-Dibutyrate; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D010769:Phosphothreonine; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D011993:Recombinant Fusion Proteins; D000099149:Ezrin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C496628:PRKCA protein, human