Activation of m-calpain (calpain II) by epidermal growth factor is limited by protein kinase A phosphorylation of m-calapin

Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Molecular and Cellular Biology (Impact Factor: 5.04). 05/2002; 22(8):2716-27. DOI: 10.1128/MCB.22.8.2716-2727.2002
Source: PubMed

ABSTRACT We have shown previously that the ELR-negative CXC chemokines interferon-inducible protein 10, monokine induced by gamma interferon, and platelet factor 4 inhibit epidermal growth factor (EGF)-induced m-calpain activation and thereby EGF-induced fibroblast cell motility (H. Shiraha, A. Glading, K. Gupta, and A. Wells, J. Cell Biol. 146:243-253, 1999). However, how this cross attenuation could be accomplished remained unknown since the molecular basis of physiological m-calpain regulation is unknown. As the initial operative attenuation signal from the CXCR3 receptor was cyclic AMP (cAMP), we verified that this second messenger blocked EGF-induced motility of fibroblasts (55% +/- 4.5% inhibition) by preventing rear release during active locomotion. EGF-induced calpain activation was inhibited by cAMP activation of protein kinase A (PKA), as the PKA inhibitors H-89 and Rp-8Br-cAMPS abrogated cAMP inhibition of both motility and calpain activation. We hypothesized that PKA might negatively modulate m-calpain in an unexpected manner by directly phosphorylating m-calpain. A mutant human large subunit of m-calpain was genetically engineered to negate a putative PKA consensus sequence in the regulatory domain III (ST369/370AA) and was expressed in NR6WT mouse fibroblasts to represent about 30% of total m-calpain in these cells. This construct was not phosphorylated by PKA in vitro while a wild-type construct was, providing proof of the principle that m-calpain can be directly phosphorylated by PKA at this site. cAMP suppressed EGF-induced calpain activity of cells overexpressing a control wild-type human m-calpain (83% +/- 3.7% inhibition) but only marginally suppressed that of cells expressing the PKA-resistant mutant human m-calpain (25% +/- 5.5% inhibition). The EGF-induced motility of the cells expressing the PKA-resistant mutant also was not inhibited by cAMP. Structural modeling revealed that new constraints resulting from phosphorylation at serine 369 would restrict domain movement and help "freeze" m-calpain in an inactive state. These data point to a novel mechanism of negative control of calpain activation, direct phosphorylation by PKA.

Download full-text


Available from: Alan Wells, Aug 10, 2015
  • Source
    • "The A-isomer has been shown to induce migration and proliferation, and the B-isomer to inhibit migration and proliferation (Lasagni et al., 2003). In fibroblasts, this receptor actuates the cAMP-PKA pathway, which results in negative phosphorylation of m-calpain to prevent growth-factorinduced motility (Shiraha et al., 2002). In keratinocytes, CXCR3 is linked to activation of PLCβ to induce a calcium-flux-mediated activation of μ-calpain enabling greater motility (Satish et al., 2005). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The signals that prune the exuberant vascular growth of tissue repair are still ill defined. We demonstrate that activation of CXC chemokine receptor 3 (CXCR3) mediates the regression of newly formed blood vessels. We present evidence that CXCR3 is expressed on newly formed vessels in vivo and in vitro. CXCR3 is expressed on vessels at days 7-21 post-wounding, and is undetectable in unwounded or healed skin. Treatment of endothelial cords with CXCL10 (IP-10), a CXCR3 ligand present during the resolving phase of wounds, either in vitro or in vivo caused dissociation even in the presence of angiogenic factors. Consistent with this, mice lacking CXCR3 express a greater number of vessels in wound tissue compared to wild-type mice. We then hypothesized that signaling from CXCR3 not only limits angiogenesis, but also compromises vessel integrity to induce regression. We found that activation of CXCR3 triggers micro-calpain activity, causing cleavage of the cytoplasmic tail of beta3 integrins at the calpain cleavage sites c'754 and c'747. IP-10 stimulation also activated caspase 3, blockage of which prevented cell death but not cord dissociation. This is the first direct evidence for an extracellular signaling mechanism through CXCR3 that causes the dissociation of newly formed blood vessels followed by cell death.
    Journal of Cell Science 07/2009; 122(Pt 12):2064-77. DOI:10.1242/jcs.048793 · 5.33 Impact Factor
  • Source
    • "Whether sumoylation at K390 contributes to an increase of cal- pain-2 binding affinity to calcium and/or phospholipids for the activation of calpain-2 activity in cells needs to be further studied. In addition, the domain III also harbors the PKA phosphorylation sites (S369 and T370) [6] "
    [Show abstract] [Hide abstract]
    ABSTRACT: Small ubiquitin-like modifier (SUMO) modification has been shown to be involved in the regulation of various cellular processes including gene transcription, nucleocytoplasmic transport, cell cycle, DNA repair, stress response, and signal transduction. However, very little is known about the process of cell migration being modulated by SUMO modification. Here, we show that calpain-2, a protease involved in cell motility, can be SUMO modified at lysine residue 390. Converting the SUMO acceptor lysine residue to arginine residue significantly attenuated calpain-2 activity, correlating well with a loss of calpain-2-elicited cell motility. Accordingly, expression of SENP1 could abrogate calpain-2 sumoylation, causing an inhibition on calpain-2-dependent activity and cell motility. These results not only identify calpain-2 as a substrate for sumoylation but also provide an important role of sumoylation in regulating cell migration.
    Biochemical and Biophysical Research Communications 06/2009; 384(4):444-9. DOI:10.1016/j.bbrc.2009.04.152 · 2.28 Impact Factor
  • Source
    • "Calpain has been shown to be involved in the migration of numerous cell types including CHO cells (Huttenlocher et al., 1997), fibroblasts (Shiraha et al., 2002), myoblasts (Leloup et al., 2006), and neutrophils (Lokuta et al., 2003). The chemokine CCL2 (previously known as monocyte chemoattractant protein-1) and its predominant receptor CCR2, a G protein–coupled receptor, have been extensively associated with inflammatory pathologies (Jee et al., 2002; Mahad et al., 2006). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Massive T cell infiltration into the central nervous system is a hallmark of multiple sclerosis (MS) and its rodent model experimental autoimmune encephalomyelitis (EAE), resulting in the induction of many of the pathophysiological events that lead to neuroinflammation and neurodegeneration. Thus, blocking T cell migration into the central nervous system may reduce disease severity in MS and EAE. One potential target for reducing T cell migration is inhibition of the Ca(2+)-activated neutral protease calpain. Previous studies in other cell types have demonstrated that migration is reduced by incubation of cells with calpain inhibitors. Thus, we hypothesize that calpain inhibition will reduce migration of T cells in response to and toward the chemokine CCL2. To test this hypothesis, the intracellular free Ca(2+) levels in Jurkat E6-1 T cells was first measured by the fura-2 assay to assess whether the intracellular ion environment would support calpain activation. The intracellular free Ca(2+) levels were found to increase in response to CCL2. The cells were next treated with the calpain inhibitor calpeptin in a multiwelled Boyden chamber with CCL2 used as the chemoattractant. These studies demonstrate that inhibition of calpain with its inhibitor calpeptin produces a dose-dependent inhibition of chemotaxis. Calpain activity, as measured by live cell imaging, was also increased in response to CCL2, providing further evidence of its involvement in the process of chemotaxis and migration. These studies provide evidence for the involvement of calpain in the mechanisms of chemotaxis and warrants further exploration in MS patient and EAE animal samples.
    Journal of Neuroscience Research 02/2009; 87(3):626-35. DOI:10.1002/jnr.21882 · 2.73 Impact Factor
Show more