The calpain cleavage sites in the epidermal growth factor receptor kinase domain

Abstract

The proteolysis of the human epidermal growth factor receptor cytoplasmic domain by calpain has been studied in vitro using purified recombinant cytoplasmic domain expressed in insect cells. Limited proteolysis produced kinase that was truncated at either N‐ or C‐termini, as well as in the hinge region. We identified seven sites of calpain proteolysis by N‐terminal sequencing of purified fragments. Calpain cleaved between the catalytic and autophosphorylation domains at two sites in the sequence Gln996–Asp1059, in the hinge region. Three new sites were also found in the autophosphorylation domain, preceding each of the major autophosphorylation sites. A fourth new site was located in the juxtamembrane domain, C‐terminal to the regulatory Thr654. We purified an active 42‐kDa fragment generated by calpain proteolysis between Leu659–Gln660 in the juxtamembrane domain, and in the hinge region. A fifth new site of calpain cleavage was found between the nucleotide binding motif Gly‐Xaa‐Gly‐Xaa‐Xaa‐Gly and the essential Lys721 in the catalytic core of the kinase. Since both of these features are required for catalysis, calpain cleavage at this site may potentially provide a mechanism for down‐regulation of kinase activity in vivo, under conditions of calpain activation. Thus the distribution of calpain cleavage sites along the kinase domain is consistent with a role for calpain both as a processing and as a degradative protease in epidermal growth factor receptor signalling.

Calpain regulates sensitivity to trastuzumab and survival in HER2-positive breast cancer

“…Finally, catalytically inactive calpain1 increases survival of cells exposed to trastuzumab. These findings together with observations that calpain is activated following the activation of EGFR/HER1 (Glading et al, 2004) and that calpain induces cleavage of the kinase domain of EGFR/HER1 (Gregoriou et al, 1994) support the role of calpain in regulation of activity and function of HER family and transmission of downstream signals.…”

“…Calpain cleaves HER2 in vitro and in vivo Calpain induces cleavage of the cytoplasmic region of EGFR/HER1, the prototype of HER family in vitro (Gregoriou et al, 1994). To investigate cleavage of HER2, glutathione-S-transferase (GST)-fusion protein of the C-terminal, cytoplasmic region ( Figure 1a, lane 1) was incubated with calpain1 alone (lane 2) or along with calpeptin, inhibitor of the catalytic site (lane 3).…”

Downregulation of epidermal growth factor receptor signaling by singlet oxygen through activation of caspase-3 and protein phosphatases

“…The net result is downregulation of the receptors on the membrane surface. In addition, receptors can also be degraded by proteasome and calpain-mediated pathways (Gregoriou et al, 1994;Longva et al, 2002).…”

The Calpain System

“…A similar cleavage occurs in vitro in embryonic mouse brain (46). EGF receptor kinase Degraded at seven sites, three of them in a "calpain-sensitive" hinge region between residues 996 and 1059 to produce a 150-kDa fragment from the 170-kDa EGF receptor kinase (144). The 150-kDa fragment retains EGF binding and EGF-stimulated kinase activity, but has reduced autophosphorylation activity.…”