Gregoriou M, Willis A C, Pearson M A, Crawford C
Department of Biochemistry, University of Oxford, England.
Eur J Biochem. 1994 Jul 15;223(2):455-64. doi: 10.1111/j.1432-1033.1994.tb19013.x.
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.
利用在昆虫细胞中表达的纯化重组胞质结构域，在体外研究了钙蛋白酶对人表皮生长因子受体胞质结构域的蛋白水解作用。有限的蛋白水解产生了在N端或C端以及铰链区被截断的激酶。通过对纯化片段进行N端测序，我们确定了钙蛋白酶的七个蛋白水解位点。钙蛋白酶在铰链区序列Gln996 - Asp1059的两个位点处，在催化结构域和自磷酸化结构域之间进行切割。在自磷酸化结构域中，在每个主要自磷酸化位点之前还发现了三个新位点。第四个新位点位于紧邻膜结构域，在调节性苏氨酸654的C端。我们纯化了一个由钙蛋白酶在紧邻膜结构域和铰链区的Leu659 - Gln660之间进行蛋白水解产生的活性42 kDa片段。在激酶催化核心的核苷酸结合基序Gly - Xaa - Gly - Xaa - Xaa - Gly和必需的赖氨酸721之间发现了钙蛋白酶切割的第五个新位点。由于这两个特征都是催化所必需的，在钙蛋白酶激活的条件下，该位点的钙蛋白酶切割可能为体内激酶活性的下调提供一种机制。因此，钙蛋白酶切割位点沿激酶结构域的分布与钙蛋白酶在表皮生长因子受体信号传导中作为加工蛋白酶和降解蛋白酶的作用是一致的。
