The calpain cleavage sites in the epidermal growth factor receptor kinase domain.
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 juxta-membrane domain, C-terminal to the regulatory Thr654. We purified an active 42-kDa fragment generated by calpain proteolysis between Leu659-Gln660 in the juxta-membrane 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.
Gregoriou
M
M
Department of Biochemistry, University of Oxford, England.
Willis
A C
AC
Pearson
M A
MA
Crawford
C
C
D000595:Amino Acid Sequence; D001345:Autoradiography; D001665:Binding Sites; D002154:Calpain / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D066246:ErbB Receptors; D002154:Calpain