Phosphoinositide Binding to the Substrate Regulates Susceptibility to Proteolysis by Calpain

Abstract

Calpain-mediated proteolysis regulates cytoskeletal dynamics and is altered during aging and the progression of numerous diseases or pathological conditions. Although several cytoskeletal proteins have been identified as substrates, how localized calpain activity is regulated and the mechanisms controlling substrate recognition are not clear. In this study, we report that phosphoinositide binding regulates the susceptibility of the cytoskeletal adhesion protein ␣-actinin to proteolysis by calpains 1 and 2. At first, ␣-actinin did not appear to be a substrate for calpain 2; however, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) binding to ␣-actinin resulted in nearly complete proteolysis of the full-length protein, producing stable breakdown products. Calpain 1 was able to cleave ␣-actinin in the absence of phosphoinositide binding; however, PtdIns(3,4,5)P 3 binding increased the rate of proteolysis, and phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P 2 ) binding significantly inhibited cleavage. Phosphoinositide binding appeared to regulate calpain proteolysis of ␣-actinin by modulating the exposure of a highly sensitive cleavage site within the calponin homology 2 domain. In U87MG glioblastoma cells, which contain elevated levels of PtdIns(3,4,5)P 3 , ␣-actinin colocalized with calpain within dynamic actin cytoskeletal structures. Furthermore, proteolysis of ␣-actinin producing stable breakdown products was observed in U87MG cells treated with calcium ionophore to activate the calcium-dependent calpains. Additional evidence of PtdIns(3,4,5)P 3 -mediated calpain proteolysis of ␣-actinin was observed in rat embryonic fibroblasts. These results suggest that PtdIns(3,4,5)P 3 binding is a critical determinant for ␣-actinin proteolysis by calpain. In conclusion, phosphoinositide binding to the substrate is a potential mechanism for regulating susceptibility to proteolysis by calpain.Calpains 1 and 2 are ubiquitous calcium-dependent proteases that play an important role in the signaling of various cellular processes and have been implicated in the degeneration observed in numerous pathological conditions (1). The requirement of calcium concentrations above physiological levels, micromolar for calpain 1 and millimolar for calpain 2, has stimulated much investigation for other factors involved in the activation of calpain. Autolysis lowers the concentration of calcium required for half-maximal activity from 7.1 to 0.6 M for calpain 1 and from 1000 to 180 M for calpain 2 (2). However, there is currently no evidence that autolysis is required for calpain modulation in cells. PtdIns(4,5)P 2 3 binding also lowers the concentration of calcium required for the activation of calpain (3-5) and is a potential mechanism for regulating calpain during cell migration (6). More recently, Glading et al. (7-9) published a series of studies showing that phosphorylation on serine 50 by extracellular signal-regulated kinase activates calpain 2, mediating detachment of the rear of the cell during epidermal growth factor-i...

Tadalafil Treatment Delays the Onset of Cardiomyopathy in Dystrophin‐Deficient Hearts

“…In these hearts, which represent a basal state (as opposed to the stressed mdx in the previous experiment) of the GRMD heart at 25 months, levels of l-calpain were relatively unchanged; in contrast, content of m-calpain, a predominantly peripheral protease, were strongly reduced with treatment ( Figure 7B). Unchanged content of the sarcomeric protein cTnI coincident with reduced cleavage products of the structural/peripheral proteins a-spectrin (150 kDa), a-actinin (80 kDa), integrin b1 (75 kDa), and dysferlin (80 kDa) [30][31][32][33] in the tadalafil-treated hearts corroborated the concept of lower levels and activity of peripheral m-calpain in these nonstimulated dystrophic hearts. We did not, however, note changes in PKG1a content or the phosphorylation status of GSK3b or ERK1/2, although a decrease in ANP and modest increases in endothelial and neuronal nitric oxide synthases were seen ( Figure S1A).…”

Regulation of the Actin Cytoskeleton-Plasma Membrane Interplay by Phosphoinositides

“…These data are supported by recent protease-sensitivity experiments, which demonstrated that PI(4,5)P 2 binding decreases the proteolysis of ␣-actinin, suggesting a role in stabilizing the structure of the protein. In contrast, PI(3,4,5)P 3 binding enhanced ␣-actinin proteolysis, indicating an increase in the flexibility of the protein (341). The in vivo importance of the interaction between ␣-actinin and PI(4,5)P 2 was demonstrated by studies on Xenopus oocytes and hippocampal neurons, where decrease in the PI(4,5)P 2 concentration or inactivation of the PI(4,5)P 2 -binding site of ␣-actinin lead to the detachement of ␣-actinin from the plasma membrane and subsequent inhibition of the NMDA receptor activity (247).…”

Integrin-dependent force transmission to the extracellular matrix by α-actinin triggers adhesion maturation

“…Second, it could adjust this force transmission through mechanisms such as the regulation of its integrin binding (56) or actin cross-linking (57) by phosphoinositides. Third, α-actinin could promote adhesion dynamics and force release by serving as a docking platform for FAK (44,58,59) or upon cleavage by calpain (60). Thus, although the specific mechanisms remain to be elucidated, these interactions could allow α-actinin to increase force generation through increased actin cross-linking and integrin binding.…”