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

Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 05/2002; 22(8):2716-27. DOI: 10.1128/MCB.22.8.2716-2727.2002
Source: PubMed


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.

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    • "The activity of Calpain-2 is negatively correlated with its phosphorylation status at a PKA consensus site [46,47]. As Nbea is an AKAP protein, Nbea haploinsufficiency might result in defects in the sequestering of inactive PKA and compartmentalization of PKA, leading to altered PKA activity in different subcellular locations [48]. "
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    ABSTRACT: Neurobeachin (NBEA) has been identified as a candidate gene for autism spectrum disorders (ASD) in several unrelated patients with alterations in the NBEA gene. The exact function of NBEA, a multidomain scaffolding protein, is currently unknown. It contains an A-kinase anchoring protein (AKAP) domain which binds the regulatory subunit of protein kinase A (PKA) thereby confining its activity to specific subcellular regions. NBEA has been implicated in post-Golgi membrane trafficking and in regulated secretion. The mechanism of regulated secretion is largely conserved between neurons and platelets, and the morphology of platelet dense granules was found to be abnormal in several ASD patients, including one with NBEA haploinsufficiency. Platelet dense granules are secreted upon vascular injury when platelets are exposed to for instance collagen. Dense granules contain serotonin, ATP and ADP, which are necessary for platelet plug formation and vascular contraction. To further investigate possible roles for NBEA in secretion or dense granule morphology, platelets from Nbea+/- mice were analyzed morphometrically, functionally and biochemically. A differential proteomics and peptidomics screen was performed between Nbea+/- and Nbea+/+ mice, in which altered Talin-1 cleavage was further investigated and validated in brain samples. Finally, the phosphorylation pattern of PKA substrates was analyzed. Platelet dense granules of Nbea+/- mice had a reduced surface area and abnormal dense-core halo, but normal serotonin-content. Nbea haploinsufficiency did not affect platelet aggregation and ATP secretion after collagen stimulation, although the platelet shape change was more pronounced. Furthermore, peptidomics revealed that Nbea+/- platelets contain significantly reduced levels of several actin-interacting peptides. Decreased levels were detected of the actin-binding head and rod domain of Talin-1, which are cleavage products of Calpain-2. This is most likely due to increased PKA-mediated phosphorylation of Calpain-2, which renders the enzyme less active. Analysis of other PKA substrates revealed both increased and reduced phosphorylation. Our results show the pleiotropic effects of alterations in PKA activity due to Nbea haploinsufficiency, highlighting the important function of the AKAP domain in Nbea in regulating and confining PKA activity. Furthermore, these results suggest a role for Nbea in remodeling the actin cytoskeleton of platelets.
    Molecular Autism 11/2013; 4(1):43. DOI:10.1186/2040-2392-4-43 · 5.41 Impact Factor
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    • "Activation of CXCR3-A has been shown to induce chemotaxis and proliferation in various cells types [42, 43]. Alternatively, CXCR3-B activation inhibits migration and proliferation and induces apoptosis [42, 44, 45]. There is emerging evidence showing that IP-10 mainly acts as an antiangiogenic factor via its signaling through CXCR3 [46, 47]. "
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    • "Activation of CXCR3-A has been shown to induce chemotaxis and proliferation in various cells types [5], [6]. Alternatively, CXCR3-B activation inhibits migration and proliferation and induces apoptosis [6], [7], [8]. In addition, IP-10 and PF4 have been reported to be angiostatic and have anti-tumor activity via its signaling through CXCR3 [7], [9], [10]. "
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