Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain

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.

Regulating cell migration: calpains make the cut

“…Cyclic-AMP-mediated activation of protein kinase A (PKA) can block EGF-induced activation of calpain 2 and fibroblast migration (Shiraha et al, 2002). This appears to occur through phosphorylation of calpain 2 by PKA, which probably restricts calpain 2 to an inactive conformation (Shiraha et al, 2002;Smith et al, 2003).…”

“…Cyclic-AMP-mediated activation of protein kinase A (PKA) can block EGF-induced activation of calpain 2 and fibroblast migration (Shiraha et al, 2002). This appears to occur through phosphorylation of calpain 2 by PKA, which probably restricts calpain 2 to an inactive conformation (Shiraha et al, 2002;Smith et al, 2003). The residues in calpain 2 (Ser369 and Thr370) that appear to be the PKA targets are conserved in other calpains, which suggests that phosphorylation of domain III represents yet another mechanism for regulating calpain activity.…”

Myoblast migration is prevented by a calpain‐dependent accumulation of MARCKS

“…Myogenic transcription factors could positively regulate this activity. On the other hand calpain activity could be also negatively regulated by phosphorylation through PKA or its endogenous inhibitor calpastatin (Melloni et al, 1984;Inomata et al, 1988;Saido et al, 1992;Croall and McGrody, 1994;Moldoveanu et al, 2001;Shiraha et al, 2002;Dedieu et al, 2003(a)). The calpain family is thought to be involved in a range of various diseases such as cataract formation, diabetes, rheumatoid arthritis, ischemia, neurodegenerative diseases and muscular dystrophies (David et al, 1993;Saito et al, 1993;Mouatt-Prigent et al, 1996;Hirsch et al, 1997;Ishikawa et al, 1999;Horikawa et al, 2000;Richard et al, 2000;Tidball and Spencer 2000;Tamada et al, 2001;Trumbeckaite et al, 2003).…”

Phosphorylation of neurofibromin by PKC is a possible molecular switch in EGF receptor signaling in neural cells

“…This dual role of EGFR in specifying cell proliferation versus motility is thought to depend on the balance between robust activation of the mitogenic Ras pathway or the concurrently activated PLC-g pathway (Xie et al, 1998), which initiates a protein kinase C (PKC)-mediated negative feedback on Ras (Chen et al, 1996). These observations have implicated PKC-protein substrates, in particular proteins involved in actin-dependent cell motility, as modulators of the biological outcome of EGFR signaling (Chen et al, 1996;Shiraha et al, 2000).…”

“…EGFRs stimulate mitosis in clonally derived, highly expandable neural stem cells and cell lines (Kornblum et al, 1999;Cheng et al, 2000a;Represa et al, 2001), regulate neural cell movement and migration (Burrows et al, 1997;Duchek and Rorth, 2001), and modulate neural cell differentiation and survival (Threadgill et al, 1995;Sibilia et al, 1998). Moreover, depending on the developmental status of the cell, the integrated biological response to EGFR-activated concurrent signaling cascades may vary widely from migratory to proliferative (Chen et al, 1996;Cheng et al, 2000a;Shiraha et al, 2000). This dual role of EGFR in specifying cell proliferation versus motility is thought to depend on the balance between robust activation of the mitogenic Ras pathway or the concurrently activated PLC-g pathway (Xie et al, 1998), which initiates a protein kinase C (PKC)-mediated negative feedback on Ras (Chen et al, 1996).…”