Epidermal growth factor activates m-calpain (calpain II), at least in part, by extracellular signal-regulated kinase-mediated phosphorylation.
How m-calpain is activated in cells has challenged investigators because in vitro activation requires near-millimolar calcium. Previously, we demonstrated that m-calpain activation by growth factors requires extracellular signal-regulated kinase (ERK); this enables tail deadhesion and allows productive motility. We now show that ERK directly phosphorylates and activates m-calpain both in vitro and in vivo. We identified serine 50 as required for epidermal growth factor (EGF)-induced calpain activation in vitro and in vivo. Replacing the serine with alanine limits activation by EGF and subsequent cell deadhesion and motility. A construct with the serine converted to glutamic acid displays constitutive activity in vivo; expression of an estrogen receptor fusion construct produces a tamoxifen-sensitive enzyme. Interestingly, EGF-induced m-calpain activation occurs in the absence of increased intracellular calcium levels; EGF triggers calpain even in the presence of intracellular calcium chelators and in calcium-free media. These data provide evidence that m-calpain can be activated through the ERK cascade via direct phosphorylation and that this activation may occur in the absence of cytosolic calcium fluxes.
Departments of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
A
A
Glading
R J
RJ
Bodnar
I J
IJ
Reynolds
H
H
Shiraha
L
L
Satish
D A
DA
Potter
H C
HC
Blair
A
A
Wells
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002465:Cell Movement; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D020935:MAP Kinase Signaling System; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry
D018076:DNA, Complementary; D011994:Recombinant Proteins; D012694:Serine; D004815:Epidermal Growth Factor; D020928:Mitogen-Activated Protein Kinases; D002154:Calpain; C449000:m-calpain; D002118:Calcium