DOI: 10.1111/j.1432-1033.1994.tb19013.x. The calpain cleavage sites in the epidermal growth factor receptor kinase domain

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

10.1111/j.1432-1033.1994.tb19013.x

Crossref journal-article

Wiley

European Journal of Biochemistry (311)

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.

Bibliography

GREGORIOU, M., WILLIS, A. C., PEARSON, M. A., & CRAWFORD, C. (1994). The calpain cleavage sites in the epidermal growth factor receptor kinase domain. European Journal of Biochemistry, 223(2), 455â464. Portico.

Authors 4

Mary GREGORIOU (first)
Anthony C. WILLIS (additional)
Mark A. PEARSON (additional)
Catherine CRAWFORD (additional)

References 54 Referenced 32

10.1038/311477a0
10.1073/pnas.81.8.2384
10.1002/jcb.240290304
10.1016/S0021-9258(18)54520-8
10.1016/0092-8674(89)90867-2
10.1016/S0021-9258(18)90895-1 / J. Biol. Chem. / Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis by Chinkers M. (1984)
10.1016/S0021-9258(19)85573-4 / J. Biol. Chem. / Epidermal growth factor‐receptor‐protein kinase interactions – co‐purification of receptor and epidermal growth factor‐enhanced phosphorylation activity by Cohen S. (1980)
10.1016/S0021-9258(19)68224-4 / J. Biol. Chem. / A native 170,000 epidermal growth‐factor receptor‐kinase complex from shed plasma‐membrane vesicles by Cohen S. (1982)
10.1016/S0021-9258(17)39032-4 / J. Biol. Chem. / Internalization of functional epidermal growth factor: receptor/kinase complexes in A‐431 cells by Cohen S. (1985)
10.1016/S0021-9258(18)48406-2
10.1042/bj2650575
10.1073/pnas.75.6.2644
10.1128/MCB.4.9.1718
10.1016/S0021-9258(18)48402-5 / J. Biol. Chem. / Epidermal growth factor (EGF)‐stimulated tyrosine phosphorylation and EGF receptor degradation in cells expressing EGF receptors truncated at residue 973 by Decker S. J. (1992)
10.1038/311483a0
10.1083/jcb.102.1.24
10.1016/S0021-9258(18)32595-X
10.1016/0092-8674(90)90474-S
10.1083/jcb.117.1.203
10.1002/pro.5560021102
10.1083/jcb.120.1.77
10.1016/S0021-9258(18)48300-7 / J. Biol. Chem. / Change in metabolic turnover is an alternate mechanism increasing cell surface epidermal growth factor receptor levels in tumor cells by Gamou S. (1987)
10.1016/0006-291X(83)90459-X
10.1021/bi00340a038
10.1002/mrd.1080270110
10.1002/j.1460-2075.1985.tb04016.x
10.1016/0092-8674(87)90147-4
10.1038/311414a0
10.1038/311480a0
10.1016/S0021-9258(19)83935-2 / J. Biol. Chem. / Epidermal growth factor receptor kinase translocation and activation in vivo by Kay D. G. (1986)
10.1002/j.1460-2075.1988.tb03171.x
10.1016/0003-9861(85)90488-6
10.1016/0167-4889(90)90160-F
10.1083/jcb.109.6.2751
10.1016/S0021-9258(18)55456-9
10.1002/j.1460-2075.1990.tb07887.x
{'key': 'e_1_2_2_38_1', 'first-page': '6170', 'article-title': 'Enhanced tumorigenesis of NR6 cells which express non‐down‐regulating epidermal growth factor receptors', 'volume': '51', 'author': 'Masui H.', 'year': '1991', 'journal-title': 'Cancer Res.'} / Cancer Res. / Enhanced tumorigenesis of NR6 cells which express non‐down‐regulating epidermal growth factor receptors by Masui H. (1991)
10.1016/S0021-9258(18)61070-1
10.1016/S0076-6879(87)46036-9
10.1016/0003-2697(87)90587-2
10.1016/S0021-9258(18)69098-2 / J. Biol. Chem. / The epidermal growth factor receptor as a substrate for a kinase‐splitting membranal proteinase by Seger R. (1988)
10.1016/0092-8674(93)90404-E
10.1126/science.8342026
10.1016/S0021-9258(18)42495-7 / J. Biol. Chem. / Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors by Sorkin A. (1992)
10.1002/jcp.1041200306
10.1083/jcb.98.3.1048
10.1002/jcb.240380106
10.1002/jcp.1041420210
10.1038/309418a0
10.1016/S0021-9258(19)40080-X
10.1038/342505a0
10.1126/science.2305263
10.1016/S0021-9258(18)99131-3
10.1016/S0021-9258(17)44660-6 / J. Biol. Chem. / Calcium‐mediated degradation of epidermal growth factor receptor in dislodged A431 cells and membrane preparations by Yeaton R. W. (1983)

Dates

Type	When
Created	20 years, 5 months ago (March 4, 2005, 5:59 a.m.)
Deposited	1 year, 9 months ago (Nov. 23, 2023, 11:07 a.m.)
Indexed	3 months, 1 week ago (May 20, 2025, 9:31 a.m.)
Issued	31 years, 1 month ago (July 1, 1994)
Published	31 years, 1 month ago (July 1, 1994)
Published Online	20 years, 5 months ago (March 3, 2005)
Published Print	31 years, 1 month ago (July 1, 1994)

Funders 0

None

BibTeX

@article{GREGORIOU_1994, title={The calpain cleavage sites in the epidermal growth factor receptor kinase domain}, volume={223}, ISSN={1432-1033}, url={http://dx.doi.org/10.1111/j.1432-1033.1994.tb19013.x}, DOI={10.1111/j.1432-1033.1994.tb19013.x}, number={2}, journal={European Journal of Biochemistry}, publisher={Wiley}, author={GREGORIOU, Mary and WILLIS, Anthony C. and PEARSON, Mark A. and CRAWFORD, Catherine}, year={1994}, month=jul, pages={455–464} }

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