Phosphoinositide Binding to the Substrate Regulates Susceptibility to Proteolysis by Calpain*

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 α-actinin to proteolysis by calpains 1 and 2. At first, α-actinin did not appear to be a substrate for calpain 2; however, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) binding to α-actinin resulted in nearly complete proteolysis of the full-length protein, producing stable breakdown products. Calpain 1 was able to cleave α-actinin in the absence of phosphoinositide binding; however, PtdIns(3,4,5)P3 binding increased the rate of proteolysis, and phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P2) binding significantly inhibited cleavage. Phosphoinositide binding appeared to regulate calpain proteolysis of α-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)P3, α-actinin colocalized with calpain within dynamic actin cytoskeletal structures. Furthermore, proteolysis of α-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)P3-mediated calpain proteolysis of α-actinin was observed in rat embryonic fibroblasts. These results suggest that PtdIns(3,4,5)P3 binding is a critical determinant for α-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)(4)(5) and is a potential mechanism for regulating calpain during cell migration (6). More recently, Glading et al. (7)(8)(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-induced fibroblast migration. Although progress has been made in understanding how calpain activity is regulated in the cell, lack of knowledge regarding the mechanisms controlling substrate recognition limits the understanding of calpain function.
Calpains 1 and 2 cleave several adhesion proteins involved in cell motility, and inhibition of calpain activity reduces cell migration (10). A role for calpain proteolysis in cell adhesion was first proposed in a 1987 study by Beckerle et al. (11), which localized calpain 2 to focal adhesions and demonstrated that talin was a sensitive substrate. In the study, the authors showed that purified ␣-actinin was not susceptible to calpain proteolysis (11). Using pharmacological inhibitors, the importance of calpain activity was extended to cell migration, identifying a role for calpain in the detachment of the rear of the cell during migration (12,13). A role for calpain in the regulation of cell migration was further established when embryonic fibroblasts lacking calpain 1 and 2 activity were observed to have decreased rates of migration correlating with a loss of actin stress fibers and focal adhesions (14). Cell adhesion and migration have become a valuable model system for examining the physiological regulation and function of calpain proteolysis.
Although the work cited above provides convincing evidence that calpains 1 and 2 are necessary for the migration of various cell types, the precise mechanisms controlling substrate recognition during cell migration are not understood. Phosphoinositides are also important for cell migration, particularly dur-ing chemotaxis (15,16). PtdIns(3,4,5)P 3 is required for establishing polarity and actin polymerization at the leading edge of migrating cells (17), and more recently, a role for PtdIns(3,4,5)P 3 was identified at the rear of migrating cells (18). PtdIns(4,5)P 2 also appears to play an important role at the leading edge and rear of migrating cells (19). Although the specific functions of PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 during cell migration are not clearly defined, both phosphoinositides bind to and regulate the structure and function of various cytoskeletal and adhesion proteins (20). Two studies have identified loose connections between phosphoinositide binding and calpain proteolysis. The first showed that overexpression of phosphoinositide 3-kinase (PI3K) enhanced calpain proteolysis of fodrin, a brain-specific isoform of spectrin (21). The second demonstrated that calpain cleaved ␣-actinin in T cells (22) stimulated under conditions that activate PI3K (23). On the basis of these studies, we proposed the hypothesis that the susceptibility of adhesion proteins to calpain proteolysis is controlled by phosphoinositide binding to the substrate protein.
To test this hypothesis, we examined the proteolysis of ␣-actinin by calpain in the absence and presence of PtdIns(4,5)P 2 or PtdIns(3,4,5)P 3 in vitro and in cultured glioblastoma cells. Our results demonstrate that phosphoinositide regulation of ␣-actinin structure controls access to the calpain cleavage site, modulating susceptibility to proteolytic post-translational modification.
Calpain Proteolysis Assays-␣-Actinin (1 M) was preincubated with phosphoinositides for 15 min at 30°C. Incubations were continued for an additional 1 h in the absence or presence of calpain and 1 mM calcium. Reactions were stopped by the addition of a modified gel loading buffer containing 250 mM Tris, pH 6.8, 2% SDS, 5 mM EGTA, 5 mM EDTA, 25 mM dithiothreitol, and 10% glycerol, followed by a 2-min incubation at 100°C. The proteins were separated by SDS-PAGE, stained with GelCode Blue (Pierce) or transferred to nitrocellulose for Western blotting, and quantified using a Kodak Image Station 440CF.
N-terminal Sequencing-Following calpain proteolysis and electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Millipore). Protein bands were identified by Ponceau S staining and excised, and N-terminal sequence analysis was carried out by the University of Texas Medical Branch Biomolecular Resource Facility (Galveston, TX).
Cell Culture and Immunofluorescence Microscopy-U87MG glioblastoma cells were cultured in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 2 mM glutamine, and 10% fetal bovine serum following the protocols from the supplier (ATCC). Immunostaining was carried out using cells plated on fibronectin as described (27) and fixed with 3% formaldehyde (Tousimis) in phosphate-buffered saline for 30 min at 25°C. Cells were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline for 5 min; blocked with 1% bovine serum albumin in phosphate-buffered saline overnight at 4°C; and incubated for 1 h at 37°C with anti-␣-actinin IgM (1:500), anti-calpain 1 IgG (1:20), or anti-calpain 2 IgG (1:20). Cells were then washed with phosphate-buffered saline, incubated for 30 min at 37°C with the appropriate fluorescein isothiocyanate-or Texas Red-labeled secondary antibody (The Jackson Laboratory, Bar Harbor, ME), and mounted using ProLong Gold antifade reagent (Invitrogen). Immunostaining was examined using a Zeiss Axiovert 100ϫ microscope, and images were captured with a Photometrics CoolSNAP HQ charge-coupled device camera controlled by Metamorph 6.3 imaging software.

PtdIns(3,4,5)P 3 Binding Increases the Susceptibility of ␣-Actinin to Proteolysis by Calpain 2-Although evidence has been
reported that calpain is involved in the proteolysis of ␣-actinin in T cells (22), ␣-actinin was shown to be a poor substrate for calpain 2 in vitro (11). However, the in vitro assays were carried out in the absence of phosphoinositides. In the presence of PtdIns(3,4,5)P 3 , ␣-actinin was highly susceptible to proteolysis by calpain 2 (Fig. 1). Consistent with the previous report (11), Ͻ20% of the ␣-actinin protein was cleaved by calpain 2 in the absence of PtdIns(3,4,5)P 3 (Fig. 1, lane 2). However, when PtdIns(3,4,5)P 3 was bound to ␣-actinin, almost complete proteolysis of the full-length protein was observed, leaving stable breakdown products of ϳ80 and ϳ65 kDa (Fig. 1, lane 4). Although differences in the size of breakdown products were observed, PtdIns(4,5)P 2 did not alter the extent of ␣-actinin proteolysis by calpain 2 (Fig. 1, lane 6). The increase in susceptibility resulting from PtdIns(3,4,5)P 3 binding to ␣-actinin was observed at low nanomolar concentrations of calpain ( Fig. 2). At a concentration of 10 nM calpain 2, the major breakdown product appeared to be ϳ80 kDa, consistent with the size of the ␣-actinin fragment resulting from the activation of calpain in anti-CD3-treated T cells (22). In addition, anti-CD3 also activates PI3K in T cells (23), further supporting the hypothesis that PtdIns(3,4,5)P 3 binding regulates the susceptibility of ␣-actinin to proteolysis by calpain. The small increase in the size of the major calpain 2-induced breakdown product observed with increasing concentrations of PtdIns(3,4,5)P 3 indicates that binding enhances the susceptibility of ␣-actinin by exposing a highly sensitive cleavage site (Fig. 3).
To determine whether PtdIns(3,4,5)P 3 influences the concentration of calcium required for calpain 2 cleavage of ␣-actinin, proteolysis assays were carried out in the presence of increasing calcium concentrations. PtdIns(3,4,5)P 3 did not appear to alter the concentration of calcium required for calpain 2 to cleave ␣-actinin (Fig. 4).
PtdIns(4,5)P 2 has been shown to bind to and influence the activity of calpain 2 (3,4); however, the presence of PtdIns(4,5)P 2 did not enhance the proteolysis of ␣-actinin by calpain 2. In addition, the presence of PtdIns(4,5)P 2 or PtdIns(3,4,5)P 3 did not affect the proteolysis of GST, a moder-ate substrate for calpain 2 (data not shown). These results support the hypothesis that it is the direct binding of PtdIns(3,4,5)P 3 to ␣-actinin that is regulating susceptibility to proteolysis by calpain.
Calpain 2 Cleavage Removes the Actin-binding Domain of ␣-Actinin-Using monoclonal antibodies specific for the N or C terminus of ␣-actinin (30), we were able to deduce that calpain 2 cleaves within the N-terminal region of the protein (Fig.  5). Immunoblotting with anti-␣-actinin clone AT6.172, recognizing the N terminus (30), demonstrated that calpain proteolysis in the presence of PtdIns(3,4,5)P 3 resulted in an almost complete lost of signal (Fig. 5A, lane 4). In contrast, immunoblotting with anti-␣-actinin clone BM-75.2, recognizing the C terminus (30), demonstrated that calpain proteolysis in the presence of PtdIns(3,4,5)P 3 resulted in a stable breakdown product of ϳ80 kDa, which included the C terminus (Fig. 5B,  lane 4). Furthermore, proteolysis of a GST fusion protein containing the actin-binding domain of ␣-actinin demonstrated that both PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 increased the susceptibility of the isolated domain to cleavage by calpain 2 (Fig.  6). However, the extent of calpain cleavage of the isolated CH2 domain was independent of phosphoinositide binding (Fig. 7). A diagram of the full-length ␣-actinin homodimer is shown in Fig. 8.
The combined results of the proteolysis assays suggest that a highly sensitive calpain cleavage site resides within the CH2    domain of ␣-actinin. The susceptibility of the CH2 domain to calpain proteolysis suggests that this cleavage site is exposed in the isolated domain (Fig. 7). However, when the actin-binding domain is expressed with the CH1 and CH2 domains together, the highly sensitive calpain cleavage site is no longer accessible. The crystal structure of the isolated ␣-actinin actin-binding domain demonstrates interaction between the CH1 and CH2 domains (31) with the potential to restrict access to the highly sensitive calpain cleavage site. Both PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 increased the susceptibility of the isolated actin-binding domain to calpain proteolysis. Presumably, phosphoinositide binding disrupted the interaction between the CH domains, increasing access to the highly sensitive calpain cleavage site. Previous studies have also presented evidence of interaction between the N-and C-terminal domains of the adjacent molecules of the ␣-actinin homodimer (32,33), which would further restrict access to the highly sensitive calpain cleavage site. Interestingly, only the binding of PtdIns(3,4,5)P 3 could expose the highly sensitive cleavage site within the ␣-actinin homodimer, increasing susceptibility to calpain proteolysis (Fig. 1). Similarly, only PtdIns(3,4,5)P 3 binding can disrupt ␣-actinin bundled actin filaments (30). As we have proposed previously, the phosphates at the fourth and fifth positions of the inositol head group of PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 appear to mediate binding with the CH2 domain, whereas the phosphate at the third position of the inositol head group appears to disrupt interaction between the N-and C-terminal domains of the ␣-actinin homodimer (25, 30).    To further understand phosphoinositide regulation of ␣-actinin proteolysis by calpain, it was important to identify the location of the highly sensitive calpain cleavage site. N-terminal sequencing of the ϳ80-kDa breakdown product resulting from calpain 2 proteolysis of PtdIns(3,4,5)P 3 -bound ␣-actinin revealed that cleavage occurred after tyrosine 246 (Fig. 8) within the final helix of the CH2 domain (31). Although the signal was significantly lower, evidence for a secondary cleavage site after serine 243 was also observed. PtdIns(3,4,5)P 3 -dependent cleavage of ␣-actinin within the CH2 domain is consistent with the binding of phosphoinositides to this domain (25).
PtdIns (3,4,5)P 3 Binding Increases and PtdIns(4,5)P 2 Binding Decreases the Susceptibility of ␣-Actinin to Proteolysis by Calpain 1-Similar to the calpain 2 isoform, calpain 1 is a ubiquitous calcium-activated protease involved in the regulation of cell migration (1,10,34). Although calpain 1 is activated by micromolar levels of calcium compared with the millimolar concentrations required for calpain 2, both isoforms appear to have the same substrate specificity. To determine whether phosphoinositide binding influences ␣-actinin susceptibility to cleavage by calpain 1, the proteolysis assay described above was repeated. Some cleavage of ␣-actinin by calpain 1 was observed in the absence of phosphoinositides (Fig. 9, lane 2). However, consistent with calpain 2, PtdIns(3,4,5)P 3 increased the susceptibility of ␣-actinin to calpain 1 proteolysis by altering the primary site of cleavage to produce a breakdown product of ϳ80 kDa (Fig. 9, lane 4). In contrast to calpain 2, PtdIns(4,5)P 2 binding inhibited calpain 1 proteolysis of ␣-actinin (Fig. 9, lane 6). N-terminal sequencing of the ϳ80-kDa breakdown product showed that calpain 1 cleaved PtdIns(3,4,5)P 3 -bound ␣-actinin after tyrosine 246, the same site as calpain 2 (Fig. 8). By exposing the cleavage site at tyrosine 246, PtdIns(3,4,5)P 3 not only increased the susceptibility of ␣-actinin but restricted calpain 1 to the production of one primary breakdown product. Thus far, the results suggest that phosphoinositide binding to the substrate influences calpain proteolysis by 1) regulating the rate or extent of proteolysis and 2) controlling the cleavage site and production of breakdown products.
To determine whether PtdIns(3,4,5)P 3 influences the concentration of calcium required for calpain 1 cleavage of ␣-acti-nin, proteolysis assays were carried out in the presence of increasing calcium concentrations. Surprisingly, the presence of PtdIns(3,4,5)P 3 increased the in vitro concentration of calcium required for calpain 1 cleavage of ␣-actinin from 125 to 250 M (Fig. 10). However, the negatively charged phosphate groups on phosphoinositides have been demonstrated to bind divalent cations in vitro (35), providing one explanation for the increased calcium requirement.
Calcium-dependent Proteolysis of ␣-Actinin in U87MG Glioblastoma Cells-Although calpain proteolysis of ␣-actinin has been shown in T cells (22) under conditions that potentially activate PI3K (23), it was important to verify these results in cells known to be regulated by PtdIns(3,4,5)P 3 . U87MG glioblastoma cells are deficient for the PtdIns(3,4,5)P 3 phosphatase PTEN and therefore have relatively high basal levels of PtdIns(3,4,5)P 3 (36,37), providing a good system to test whether ␣-actinin is a substrate for calpain in adherent cells. Because colocalization is required for an enzyme to act upon its substrate in a cellular system, we used immunofluorescence microscopy to determine the localization of ␣-actinin and calpains 1 and 2 in U87MG glioblastoma cells induced to migrate by plating on fibronectin as described previously (27). Strong staining for calpain 1 was observed in the perinuclear region of U87MG cells (Fig. 11A). However, a distinct population of calpain 1 was also observed at the leading edge of this migrating cell (Fig. 11A, arrow). ␣-Actinin staining was observed in the perinuclear region and within the adhesion complexes at the leading edge and rear of the cell (Fig. 11AЈ). A beaded staining pattern was also observed, representing the population of ␣-actinin localized along actin stress fibers. Most importantly, all of the staining observed for calpain 1 at the leading edge of the cell colocalized with ␣-actinin-containing adhesion complexes (Fig. 11A, arrow). However, not all ␣-actinin staining adhesion complexes costained for calpain 1. For example, calpain 1 staining was not observed in the ␣-actinin-containing adhesion complex at the rear of the cell (Fig. 11A, arrowhead). Similar to calpain 1, the strongest staining for calpain 2 was observed in the perinuclear region (Fig. 11B). However, distinct populations of calpain 2 staining were also observed to colocalize with  ␣-actinin within membrane ruffles at the cell edge (Fig. 11B,  arrow). These results demonstrate that calpains 1 and 2 colocalize with ␣-actinin within highly dynamic actin cytoskeletal structures of U87MG glioblastoma cells. However, the percent of the total ␣-actinin population localized to these dynamic structures is small, suggesting that only a small fraction of the total ␣-actinin population is subject to proteolysis by calpain.
Protein from U87MG glioblastoma total cell lysates was immunoblotted to assay for ␣-actinin proteolysis. The calcium ionophore A23187 was added to the cells to rapidly increase the intracellular concentrations of calcium and to activate calpain. Activation of the calcium-dependent calpain proteases was verified by immunoblotting for talin, an established calpain substrate and adhesion protein (11,38). As expected, a time-dependent increase in the previously reported talin breakdown product (11,38) was observed following treatment with A23187 (Fig. 12). Similar to talin, a time-dependent increase in ␣-actinin breakdown products was observed in the A23187-treated cells. In addition, ␣-actinin breakdown products were observed in untreated cells (t ϭ 0), suggesting that a basal level of calpain activity exists in U87MG cells. Interestingly, no talin proteolysis was observed in the untreated cells. Franco et al. (39) have reported that calpain 2 is responsible for the proteolysis of talin in fibroblasts. Therefore, it is possible that calpain 2 was responsible for the A23187-stimulated proteolysis of ␣-actinin and talin in the U87MG cells, whereas calpain 1 was responsible for the proteolysis of ␣-actinin observed in the untreated cells (Fig. 12).
PDGF-induced Proteolysis of ␣-Actinin in Fibroblasts-Numerous studies have reported that PDGF induces cell motility in a PI3K-dependent manner (40). Previously, we demonstrated that PDGF treatment of fibroblasts induced PtdIns(3,4,5)P 3 binding to ␣-actinin, resulting in the restructuring of focal adhesion plaques (28). Lysates from PDGFtreated fibroblasts were immunoblotted to determine whether ␣-actinin proteolysis correlated with focal adhesion restructuring. Time-dependent increases in ␣-actinin breakdown products were observed following 10-and 30-min treatments with PDGF (Fig. 13). The breakdown products migrated as a doublet of ϳ80 kDa, consistent with that observed for ␣-actinin proteolysis by calpain in vitro (Figs. 1-3 and 9). These results suggest that PtdIns(3,4,5)P 3 -mediated calpain proteolysis of ␣-actinin may play a role in the regulation of cell adhesion during migration.
Phosphoinositide Binding Regulates the Susceptibility of ␣-Actinin to Proteolysis in Fibroblasts-To test the hypothesis that PtdIns(3,4,5)P 3 binding regulates the susceptibility of ␣-actinin to calpain proteolysis in a cellular system, constitutively active PI3K was coexpressed with GFP-tagged wild-type or mutant ␣-actinin with a decreased affinity for phosphoinositides (25,29). Coexpression of GFP-tagged wild-type ␣-actinin with constitutively active PI3K resulted in an increase in ␣-actinin breakdown products (Fig. 14, lanes 1 and 2). In con-   . PDGF induces proteolysis of ␣-actinin in fibroblasts. Total cell lysates from PDGF-treated (30 ng/ml) fibroblasts were immunoblotted for ␣-actinin. The signal from the full-length ␣-actinin protein was so strong that to clearly image the breakdown products the immunoblot was cut, and the portion containing the uncleaved protein was developed separately from the cleavage products. Results are representative of two separate experiments. trast, an increase in ␣-actinin breakdown products was not observed when GFP-tagged mutant ␣-actinin was coexpressed with constitutively active PI3K (Fig. 14, lanes 4 and 5). Coexpression of GFP-tagged ␣-actinin with constitutively active PI3K was confirmed by immunostaining as shown in Ref. 29. Previously, we used this experimental system to demonstrate that PtdIns(3,4,5)P 3 binding to ␣-actinin played a role in regulating the disassembly of focal adhesions and the reorganization of the actin cytoskeleton (29). Results of the current study suggest that the mechanism by which PtdIns(3,4,5)P 3 binding mediates ␣-actinin function involves regulation of susceptibility to proteolysis by calpain.
The requirement for calpain activity during cell adhesion and migration is well established with influence on the processes of cell spreading, membrane protrusion, adhesion complex turnover, and tail retraction (12,38,39,(41)(42)(43)(44). In addition to ␣-actinin, talin, vinculin, filamin, spectrin, and ezrin are involved in the formation, maintenance, and turnover of adhesion complexes and are regulated by phosphoinositide binding (20,45). Interestingly, all of these adhesion proteins are also substrates for calpain (10, 22, 41, 46 -49). Although calpain cleavage of adhesion proteins is an important regulatory mechanism, it is not clear how the localization, activation, and substrate selection for calpains 1 and 2 are modulated during cell adhesion and migration. This study addresses the cellular regulation of the calpain system from the point of view of the substrate, identifying a role for phosphoinositide binding in modulating the susceptibility of ␣-actinin to calpain proteolysis. Further studies are necessary to understand the role of PtdIns(3,4,5)P 3 -regulated proteolysis of ␣-actinin by calpain in cell adhesion and migration. In addition, it will be important to determine whether calpain proteolysis of other proteins is influenced by phosphoinositide binding.