[Ubiquitination of EGF receptors with C-terminal domain deletion and point mutations during endocytosis].

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 ubiquitin. 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 ubiquitination 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.

[Remodeling of microtubule system during EGF receptor endocytosis].

The idea of microtubules (MTs) as of passive railway tracks, along which transport vesicles travel by use of motor proteins, is widely accepted. In the present work the organization of MT system during EGF-receptor endocytosis was investigated by indirect double immunofluorescence in HeLa and A431 cell lines. Stimulation of cells with EGF resulted in formation of EGF receptor-containing peripheral vesicular endosomes. During time course of endocytosis the endosomes tended to concentrate in juxtranuclear region close to MTOC. This translocation was dependent on MTs since nocodazole treatment resulted in endosomes' scattering throughout the cytoplasm. Parallel staining of the cells with tubulin antibody has revealed significant remodeling of MTs organization during endocytosis. At early stages MTs demonstrated slight retraction at the cell periphery and the increasing intensity of tubulin fluorescence in the juxtranuclear region. Later on, long individual MTs disappeared and peripheral cytoplasm show diffuse staining in combination with a meshwork of short MT fragments. This stage correlated with EGFR localization in juxtranuclear endosomes. Disappearance of EGFR-positive staining due to its lysosomal degradation occurred in parallel to reestablishment of radial MT system. Possible functional significance of described alterations in organization of tubulin cytoskeleton is discussed.

[Effect of synthetic proteasomal inhibitor MG132 on dynamics of EGF-receptor complexes endocytosis in A431 cells]. / Vliianie sinteticheskogo ingibitora proteasom MG132 na dinamiku éndotsitoza EFR-retseptornykh kompleksov v kletkakh A431.

The effect of proteasomal activity suppression induced by MG132, a synthetic proteasomal inhibitor of EGF-receptor complexes endocytosis in human epidermoid carcinoma A431 cell line, was studied. Using subcellular fractionation in 17% Percoll gradient, it was demonstrated that the addition of MG132 to the cells 15 min following stimulation of EGF endocytosis resulted in a slight accumulation of 125I-EGF in early endosomes, and in much more significant accumulation of the labeled growth factor in late endosomes/lysosomes, as compared to untreated cells. The release of 125I-EGF degradation products into the incubation medium was significantly (3-12-fold) inhibited in the presence of MG132. At the same time biochemical analysis has demonstrated that the EGF receptor itself is not a direct target of proteasomes, since it is revealed as a full-length protein with native mol. mass (170 kDa) in fractions of early and late endosomes and lysosomes. Possible mechanisms of the MG132 effect on intracellular processing of EGF-receptor complexes are discussed.

[The role of phosphatidylinositol-3-kinases P85/P110 and hVPS34 in endocytosis of EGF-receptor complexes]. / Rol' fosfatidilinozitol-3-kinaz P85/P110 i hVPS34 v éndotsitoze EFR-retseptornykh kompleksov.

The previous data (Zheleznova et al., 2001) did not enable the authors to conclude which particular wortmannin sensitive PI-3-kinase--p85/p110 (I class PI-3-K) or hVPS34 (III class PI-3-K)--may be involved in the regulation of EGF-receptor endocytosis. In the present work, we have shown that upon stimulation of EGF-receptor endocytosis additional structures stained with antibody against p85 appear in A431 cells, but the p85-positive compartment never co-localized with EGF-receptor-containing compartments either in control or in wortmannin-treated cells. At the same time, wortmannin treatment prevented association of hVPS34 with endosomal membranes. We have also found that early endosomal markers--Rab5 and EEA1 (membrane association of the latter depends on Rab5 and hVPS34)--co-localized with EGF-receptor in the juxtranuclear region during late stages of endocytosis, both in control and upon wortmannin treatment. These observations favor our suggestions that the transition of EGF-receptors from early to late endosomes may occur directly in this juxtranuclear region and be tightly associated with the formation of so called multivesicular bodies (MVB), which are late endosomes per se. We suggest that wortmannin may have no effect on early EEA1-dependent stage of the receptor endocytosis but blocks a transition of EGF-receptor complexes into the late endosomes by inhibiting activity of hVPS34 and removing it from membranes. The hVPS34 product PI-3-K, according to the known data, is involved in the formation of internal vesicles of MVB. Accumulation of EGF-receptors in these vesicles is believed to be necessary for the receptor degradation.

[Cbl--a polyfunctional regulator of cellular processes]. / Cbl--polifunktsional'nyi reguliator kletochnykh protsessov.

C-Cb1 protein is a protooncogene product that was initially identified as part of a murine retrovirus transforming protein. C-Cb1 is ubiquitously expressed in cells of different origin. A number of isoforms subsequently identified in vertebrates and invertebrates allows to consider the existence of a family of Cb1 proteins. These proteins contain a set of sequences providing interactions with a wide range of receptor and nonreceptor tyrosine kinases and signaling proteins with SH2- and SH3-domains (for example, EGF and PDGF receptors, Src-kinases, PI-3-kinase p85, Crk, GRB2, Vav, etc.). Cb1 proteins possess also multiple tyrosine, residues, which undergo phosphorylation upon stimulation of several surface receptors. These properties permit Cb1 to take part in many protein-protein interactions as an adaptor, which forms multimolecular signaling complexes, and coordinates the activity of its components. C-Cb1 and its mutant transforming forms can act as both positive and negative regulators of many signaling pathways. Negative action of C-Cb1 on signals stimulated by receptor tyrosine kinases is thought to result from accelerated receptor degradation caused by Cb1. This ability is attributed mostly to ubiquitin-ligase activity of Cb1 proteins, since the latest research evidence suggests that ubiquitination may be a signal of not only proteasomal, but also lysosomal degradation. Thus, Cb1 manifests itself as a many-sided protein working both as an adaptor and a regulator of endocytic trafficking. In spite of numerous studies in this area, the regulation of Cb1 functions, interrelations between these functions, physiological significance of Cb1-mediated interactions, and the place of Cb1 proteins in signaling coordinating still remain obscure. In the present review, an attempt is made to summarize the recent data, with special reference to Cb1 functioning as a regulator of tyrosine kinase receptor endocytosis.

[Effect of an vacuolar proton pump inhibitor bafilomycin A1 on the intracellular processing of markers for receptor-mediated and liquid phase endocytosis]. / Vliianie ingibitora vakuoliarnoi protonnoi pompy bafilomitsina A1 na vnutrikletochnyi protsessing markerov retseptor-oposredovannogo i zhidkofaznogo éndotsitoza.

By means of subcellular fractionation in density Percoll gradients, immunoblotting and immunofluorescense, the effect of BafA1 on endocytosis of EGF-receptor complexes and horse radish peroxidase (HRP) in A431, HER14 and HC11 cell lines was studied. It was shown that the pretreatment of all used cell lines with BafA1 completely inhibited EGF degradation, but did not interfere with the delivery of significant portion of EGF-receptor complexes to late endosomes and lysosomes and transition of the receptor to juxtranuclear region. At the same time, BafA1 was found to dramatically inhibit the delivery of fluid phase marker HRP to late endosomes of A431 cells. The BafA1 effect on endocytosis of high concentrations of EGF was similar to that on HRP endocytosis. Regulatory mechanisms of early-to-late endosomal compartment transition are discussed.

[The role of Src-kinase in the regulation of endocytosis of EGF-receptor complexes. I. Dynamics of EGF internalization, recycling, sorting, and degradation during inhibition of Src-kinase activity]. / Issledovanie roli Src-kinazy v reguliatsii éndotsitoza EFR-retseptornykh kompleksov. I. Analiz dinamiki internalizatsii, retsiklirovaniia, sortirovki i degradatsii EFR v usloviiakh podavleniia aktivnosti Src-kinazy.

In the present work, the role of Src-kinase in regulation of different stages of EGF-receptor endocytosis was studied. We used murine fibroblasts with knockout of Src gene and CGP77675, and the inhibitor of Src-family kinases. The absence of Src protein in the cells did not lead to any changes in the rates of 125I-EGF internalization or recycling and caused only slight decrease in the rate of its degradation. At the same time, treatment of the wild type cells with the inhibitor resulted in a small decrease in internalization rate and an increase in recycling. The influence of the inhibitor on 125I-EGF degradation was also more pronounced. But even in this case, the effects were no more than 30% of control values. CGP77675 extended the same effect upon cells of HER14 and HC11 lines. Subcellular fractionation of these cells in Percoll gradient has also demonstrated a slight inhibition of 125I-EGF sorting from early to late endosomes. The more pronounced effect of the Src-family kinase inhibitor on the EGF endocytosis, compared to that of the absence of a single Src protein, suggests a compensating mechanism of the Src-family kinases. A conclusion is made that in spite of a slight influence on practically all stages of intracellular pathway of EGF-receptor complexes, Src-kinases are obviously not the key regulators of their endocytosis.

[The role of Src-kinase in the regulation of endocytosis of EGF-receptor complexes. Distribution of clathrin after stimulation of EGR endocytosis in various cell lines during inhibition of Src-kinase activity]. / Issledovanie roli Src-kinazy v reguliatsii éndotsitoza EFR-retseptornykh kompleksov. II. Analiz raspredeleniia klatrina posle stimuliatsii éndotsitoza EFR v kletkakh razlichnykh linii v usloviiakh podavleniia aktivnosti Src-kinaz.

A distribution of EGF receptor and clathrin during EGF endocytosis in A431, HER14, WT and PURO cell lines was studied by indirect immunofluorescence. Though the initial distribution of EGF-receptors on A431 and HER14 cells was somewhat different, the late stages of endocytosis proceeded equally and were marked by formation of bright spots in the juxtanuclear region characteristic of the late endosomes. The Src-family kinase inhibitor CGP77675 had no influence on the dynamics of receptor endocytosis at the immunofluorescent level in both cell lines. Stimulation of EGF-receptor endocytosis in A431 cells did not also result in any redistribution of clathrin in the areas where the majority of EGF-receptors are localized, i.e. in the lateral plasma membrane both in the control cells and under CGP77675 treatment. Clathrin in A431, WT and PURO cells demonstrated even a punctuated pattern throughout the cytoplasm with some accumulation in the juxtanuclear region. This distribution depended neither on the absence or presence of Src activity nor on EGF addition. The data obtained indicate that 1) EGF-receptors do not serve as the initiation sites during clathrin coated pit assembly; 2) Src-kinase activation does not result in significant clathrin redistribution in the plasma membrane, and its influence on EGF endocytosis can be considered as a secondary effect.

[Endocytosis of EGF-receptor complexes at various cell cycle stages]. / Issledovanie éndotsitoza EFR-retseptornykh kompleksov na raznykh stadiiakh kletochnogo tsikla.

Parameters of EGF-receptor complex endocytosis have been studied in the early and late G1 phase and in mitosis. As a model, mouse mammary epithelial cells HC11 were used, whose growth depends on EGF presence in the medium. The Scatchard analysis has demonstrated that the surface receptors are represented by two receptor populations: 4800 high affinity (KD = 10(-11) M) receptors, and 73,000 low affinity (KD = 4.10(-9) M) receptors. Incubation of cells with the growth factor (5 ng/ml) resulted in a decrease in 125I-EGF binding, with its level being low until entering the S-phase. Under these conditions, receptors disposed on the plasma membrane presented a homogeneous population (KD = 8.10(-11) M, 14,000 receptors per cell). No reliable difference was revealed between the EGF-receptor complexes, internalized in early and late G1 phases, in respect to the internalization rate, level of recycling, degradation, and dynamics of compartmentalization. However, endocytosis of EGF-receptor complexes was found to be completely blocked in mitosis at the stage of internalization.