We present data demonstrating a novel mechanism for negative regulation of EGF-induced m-calpain activation--direct phosphorylation by PKA. We previously reported that EGF-induced m-calpain activation is negatively regulated by ELR-negative CXC chemokines (42
). However, the molecular mechanism for this regulation was undefined. Herein, we report that PKA phosphorylation of m-calpain at amino acids S369 and T370 blocks EGFR-mediated activation in vivo. The addition of a negatively charged side chain is modeled to “freeze” m-calpain in an inactive state. These findings support an unexpected model of m-calpain regulation that involves protein phosphorylation of the regulatory domain.
In brief, mutation of a putative consensus PKA site at amino acids S369 and T370 to alanines generated a calpain molecule that was resistant to CPT-cAMPS negative attenuation of EGF-induced calpain activation and cell motility. One concern about mutagenesis is that it might render the target molecule inactive. We do not feel that our failure to attenuate EGFR signaling is due to such a false-positive result since the ST369AA mutant calpain remains activable by EGF. The in vitro activity of the ST369AA mutant was similar to that of wild-type m-calpain as determined by two different calpain activity assays, cleavage of Tau and cleavage of MAP2 (Fig. ). Our other biologic reporter assay, the BOC-LM-CMAC assay, relies on the mutant calpain being activable in the face of normally negative regulation. This finding is buttressed by the fact that two different tagged constructs acted indistinguishably, expressed either transiently (single-cell calpain activation [data not shown]) or stably.
A second caveat is that we have not mapped the PKA phosphorylation site directly. This was not attempted either in vitro or in vivo. In vitro, purified m-calpain is multiply phosphorylated on both serines and threonines even in its nonactivated state (data not shown; Cong et al., Abstr. Am. Soc. Cell Biol. 40th Annu. Meet., 2000). In addition, nonphysiological conservative replacement of serine 369 could easily shift the PKA phosphorylation to the adjacent threonine 370. In vivo, the multiple, seemingly cotranslational phosphorylation would confound attempts to metabolically label m-calpain. Furthermore, as EGF activates on the small fraction of m-calpain that is in the plasma membrane (18
), we are likely dealing with a substantially substoichiometric and potentially short-lived modification. However, in our preliminary study, we could detect low-level phosphorylation of wild-type hCANP caused by PKA activator CPT-cAMPS stimulation, but not in ST369AA hCANP (data not shown). As J. Cong et al. (Abstr. Am. Soc. Cell Biol. 40th Annu. Meet., 2000) reported, calpain can be phosphorylated even in a nonactivated state; the elevation in signal above the background phosphorylation of hCANP appeared to be less than twofold. We speculate that only a low level of phosphorylation is noted because PKA phosphorylation is a regulatory event that in vivo may both only involve a small fraction of total cell m-calpain and be transient or lead to rapid m-calpain degradation. Nevertheless, PKA will phosphorylate wild-type but not ST369AA m-calpain both in vitro and in vivo, strongly suggesting that this is the target site. Additionally, the MAP2 cleavage assay data (Fig. ) demonstrate that removal of the PKA target site renders m-calpain resistant to PKA. Rather, we hypothesized that PKA directly phosphorylates the putative consensus site in the proposed regulatory domain III (25
). Alteration of this site by replacement with alanines yields a construct resistant to both PKA phosphorylation and enzymatic repression in vitro and CPT-cAMPS attenuation in vivo. These findings demonstrate that PKA phosphorylation of domain III prevents activation of m-calpain and strongly support the structure-based prediction that domain III serves as a regulatory domain (46
A third caveat is that calpain modulation may also occur via actions on proteins other than m-calpain. cAMP-dependent PKA also has been reported elsewhere to phosphorylate calpastatin (32
), the endogenous calpain inhibitor, with this phosphorylation affecting the distribution of calpastatin in neuroblastoma cells (3
). We do not exclude the possibility, implied in these reports, that phosphorylation of calpastatin by PKA might affect the regulation of calpain. However, our hypothesis that PKA direct phosphorylation of calpain inhibits calpain activation was sufficiently verified by both in vitro and in vivo experiments. These findings herein strongly suggest that direct phosphorylation of m-calpain is the major regulatory mechanism preventing EGF-induced m-calpain activation in fibroblasts. Another study reports that in vitro serine phosphorylation of bovine m-calpain, at the equivalent of S369, by calmodulin-dependent protein kinase II increases general calpain activity (37
). The relevance of this finding to our report is uncertain for two reasons. First, CaM kinase II phosphorylated only autoproteolyzed m-calpain and had no effect on full-length calpain; this is interesting in light of full-length calpain now being considered to be as active as the autolyzed form, which might simply be an intermediary of the degradative attenuation process (28
). Second, this report examined calpain activity only in vitro in the presence of supraphysiological concentrations of the activator calcium, whereas our studies were performed under cytosolic calcium concentrations in vivo; it is conceivable that opposite effects of phosphorylation at identical sites could be seen under such diverse circumstances.
Computer modeling of the phosphorylation at S369 supports our in vivo findings. We chose to focus on S369 since this is the best consensus PKA site and has been reported elsewhere to be phosphorylated as determined by phosphopeptide mapping (J. Cong et al., Abstr. Am. Soc. Cell Biol. 40th Annu. Meet., 2000); T370 was also mutated to prevent possibly nonphysiological usage of an alternate phosphorylation acceptor. Without doubt, the multiple interactions enabled by S369-P add new constraints in the interface between domain III and domain IV. In the proposed activation mode of calpain upon addition of calcium, various domains of calpain would undergo domain movement in the process of assembling the active site (22
). Experimental evidence supporting this hypothesis has been recently reported (23
), where disruption of critical interdomain constraints resulted in an increase in calcium sensitivity. By the same reasoning, if the extra constraints were added then opposite effects would occur. In the case of calpain 3 (or p94), a thorough structural analysis has again revealed that the effect on interdomain movement is crucial for the activity (26
). In the case of S369-P, S369 of domain III is strategically located at the interface between domains III and IV. Phosphorylation of S369 not only provides a highly charged group but, more importantly, “extends” the length of the side chain. Thus, it enables interactions with a couple of residues of domain IV. As a consequence, these interactions give rise to extra constraints in the interface, thereby severely restricting the freedom of both domains. The PKA consensus sequence RRxS369
is present only in the closely related μ-calpain (calpain I) and the testis-specific calpain 11 (45
). However, it remains to be demonstrated experimentally whether this other ubiquitous calpain is negatively attenuated by PKA since only R628 but not H643 is present for cross-bridging in μ-calpain. Parenthetically, the supraphysiological concentrations of calcium used to demonstrate CaM kinase II-induced calpain activity (37
) might either overcome this movement restriction or disrupt salt bridging. The rigidification imposed by phosphorylation of S369 would essentially hamper the movement of these domains in the assembly of the active site and result in the loss of activity. This modeling, by being theoretical like all modeling, provides a potential molecular basis for the inhibitory action of PKA and forwards predictions for both m-calpain structure and dominance of inhibitory signals that might guide future experimental studies that lie beyond the scope of the present work.
Stable transfections yielded only a fractional increase in total calpain levels. This was not surprising. Initially, we attempted overexpression of both wild-type and ST369AA calpains using the strong CMV promoter. In transient transfections, robust GFP fluorescence was noted shortly after electroporation, but most of the cells rounded and detached within 24 h (data not shown). We did not pursue whether this was due to calpain-mediated deadhesion (6
) or actual apoptosis as it lay beyond the scope of the present study. Calpain has been implicated elsewhere in some mechanisms of apoptosis (43
), and excess calpain activity might trigger caspase-mediated apoptosis (38
). We established NR6 cell sublines containing the MMTV-driven calpain constructs. Even in the presence of dexamethasone, we attained exogenous expression at only ~30% of m-calpain. That this was sufficient to transmit EGFR-mediated calpain activity and motility in the presence of CPT-cAMPS suggests that endogenous calpain levels are in excess of those needed for robust deadhesion during motility. This is consistent with our ancillary studies that find that EGFR-mediated deadhesion requires only the submembrane subset of m-calpain (18
In our previous paper, we presented evidence that the counterregulatory ELR-negative CXC chemokines inhibit EGF-induced cell migration but not proliferation (42
). Herein, we demonstrate that this occurs via direct PKA phosphorylation of m-calpain. This provides for testable hypotheses concerning fibroblast functioning during wound repair. In the inflammatory and reparative stages the high levels of EGFR ligands in the wound bed would promote repopulation through both motility and mitogenesis. Later in the resolution phase, the presence of IP-10 from ingrowing endothelial cells (19
) and a related CXCR3-binding chemokine from basal keratinocytes (IP-9 or I-TAC) (9
) would channel the motile phenotype to matrix contraction (2
). As cAMP has been shown elsewhere to be antiproliferative in fibroblasts (10
), a second PKA-mediated pathway would limit fibroplasia.