UBIQUITINATION OF EGF RECEPTORS WITH C-TERMINAL DOMAIN DELETION AND POINT MUTATIONS DURING ENDOCYTOSIS
K. A. Kondratov,1 M. S. Melikova,1 A. L. Chernorudsky,1, 2 E. S. Kornilova 1, 3
1 Institute of Cytology RAS, St. Petersburg, and 2 Nizhny Novgorod State Medical Academy;
3 e-mail: elena.kornilova@gmail.com
Analysis of ubiquitination of EGF receptor carrying different mutations of C-terminal domain was done. The mutants differed both by the set of major
autophosphorylation sites that determine the way of interaction with ubiquitin-ligase c-Cbl, and by the presence of lysine residues which can be possible acceptor
sites for ubi-quitin. It was found that the receptor lacking tyrosine kinase activity due to lysine for phenylalanine substitution at ATP-binding site of tyrosine kinase
(TK) domain (K721) failed to be ubiquitinated as well as the receptor without all binding sites for c-Cbl (CD165), while dynamics and pattern of ubiquitination of other
deletion mutants was significantly different. The mutant lacking Grb2 binding sites but able to bind c-Cbl directly (CD123) was minimally ubiquitinated and only at
early stages upon EGF endocytosis stimulation. At the same time, the receptor possessing all binding sites for Cbl but lacking C-terminal domain of 63 aminoacid
residues (CD63) which contains two autophosphorylation sites (Y1148 and 1173) and 4 lysines, was less ubiquitinated and had more low-ubiquitinated forms
comparing to the WT one. However, these lysines are not acceptor sites for ubiquitin since the full-size receptor lacking like CD63 the same major
autophosphorylation sites underwent ubiquitinati-on similar to the deletion mutant. Thus, C-terminal region of the EGF receptor, being not a substrate for
ubiquitination per se, is involved in its regulation. It was also found that ubiquitination pattern at fast endocytosis differed from those at slow one. In the first case
the total level of EGFR decreased dramatically as a result of efficient lysosomal degradation. The level of receptor-associated c-Cbl was practically the same, while
the total intracellular c-Cbl dropped. Treatment of cells with proteasomal inhibition MG132 blocked the loss of Cbl only partially. In the second case, total amount of
both EGF receptor and c-Cbl did not notably change that suggested recycling pathway for receptors even despite them beeng ubiquitinated.
Key words: EGF receptor, autophosphorylation sites, C-terminal domain ubiquitination, ubiquitin-ligase c-Cbl, endocytosis
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