m-Calpain activation is regulated by its membrane localization and by its binding to phosphatidylinositol 4,5-bisphosphate.
m-Calpain plays a critical role in cell migration enabling rear de-adhesion of adherent cells by cleaving structural components of the adhesion plaques. Growth factors and chemokines regulate keratinocyte, fibroblast, and endothelial cell migration by modulating m-calpain activity. Growth factor receptors activate m-calpain secondary to phosphorylation on serine 50 by ERK. Concurrently, activated m-calpain is localized to its inner membrane milieu by binding to phosphatidylinositol 4,5-bisphosphate (PIP(2)). Opposing this, CXCR3 ligands inhibit cell migration by blocking m-calpain activity secondary to a PKA-mediated phosphorylation in the C2-like domain. The failure of m-calpain activation in the absence of PIP(2) points to a key regulatory role, although whether this PIP(2)-mediated membrane localization is regulatory for m-calpain activity or merely serves as a docking site for ERK phosphorylation is uncertain. Herein, we report the effects of two CXCR3 ligands, CXCL11/IP-9/I-TAC and CXCL10/IP-10, on the EGF- and VEGF-induced redistribution of m-calpain in human fibroblasts and endothelial cells. The two chemokines block the tail retraction and, thus, the migration within minutes, preventing and reverting growth factor-induced relocalization of m-calpain to the plasma membrane of the cells. PKA phosphorylation of m-calpain blocks the binding of the protease to PIP(2). Unexpectedly, we found that this was due to membrane anchorage itself and not merely serine 50 phosphorylation, as the farnesylation-induced anchorage of m-calpain triggers a strong activation of this protease, leading notably to an increased cell death. Moreover, the ERK and PKA phosphorylations have no effect on this membrane-anchored m-calpain. However, the presence of PIP(2) is still required for the activation of the anchored m-calpain. In conclusion, we describe a novel mechanism of m-calpain activation by interaction with the plasma membrane and PIP(2) specifically, this phosphoinositide acting as a cofactor for the enzyme. The phosphorylation of m-calpain by ERK and PKA by growth factors and chemokines, respectively, act in cells to regulate the enzyme only indirectly by controlling its redistribution.
Leloup
Ludovic
L
From the Departments of Pathology, Pittsburgh, Pennsylvania 15261.
Shao
Hanshuang
H
From the Departments of Pathology, Pittsburgh, Pennsylvania 15261.
Bae
Yong Ho
YH
Bioengineering, Pittsburgh, Pennsylvania 15261.
Deasy
Bridget
B
Orthopedic Surgery, Pittsburgh, Pennsylvania 15261.
Stolz
Donna
D
Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
Roy
Partha
P
From the Departments of Pathology, Pittsburgh, Pennsylvania 15261; Bioengineering, Pittsburgh, Pennsylvania 15261.
Wells
Alan
A
From the Departments of Pathology, Pittsburgh, Pennsylvania 15261; Bioengineering, Pittsburgh, Pennsylvania 15261; Pittsburgh Veterans Affairs Medical Center, Pittsburgh, Pennsylvania 15261. Electronic address: wellsa@upmc.edu.
D000818:Animals; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D054357:Chemokine CXCL10 / Q000235:genetics / Q000378:metabolism; D054371:Chemokine CXCL11 / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation / Q000502:physiology; D017434:Protein Structure, Tertiary; D054367:Receptors, CXCR3 / Q000235:genetics / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
C114753:CXCL10 protein, human; C515202:CXCL11 protein, human; C515187:CXCR3 protein, human; D054357:Chemokine CXCL10; D054371:Chemokine CXCL11; C485433:Cxcl10 protein, mouse; C515203:Cxcl11 protein, mouse; C515188:Cxcr3 protein, mouse; D007295:Inositol Phosphates; D054367:Receptors, CXCR3; D017978:Receptors, Growth Factor; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D004815:Epidermal Growth Factor; C066454:inositol 4,5-bisphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D002154:Calpain; C449000:m-calpain