B Vynnytska, Ya Bobak, O Basaraba - Distribution of adaptor protein cin85ruk multiple molecular forms between cytosolic and nuclear fractions in human lung adenocarcinoma cell line - страница 1

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ВІСНИК ЛЬВІВ. УН-ТУ VISNYK OF L'VIV UNIV.

Серія біологічна. 2007. Вип. 43. С. 86-90 Biology series. 2007. Is. 43. P. 86-90

Біохімія

УДК 577.29

DISTRIBUTION OF ADAPTOR PROTEIN CIN85/RUK MULTIPLE MOLECULAR FORMS BETWEEN CYTOSOLIC AND NUCLEAR FRACTIONS IN HUMAN LUNG ADENOCARCINOMA CELL LINE A549

B. Vynnytska**, Ya. Bobak*, O. Basaraba*, N. Ihumentseva*, L. Drobot*

Institute of Cell Biology, National Academy of Sciences of Ukraine Drahomanov st. 14/16, UA-79005 Lviv, Ukraine Ivan Franko National University of Lviv Hrushevski st. 4, UA-79005 Lviv, Ukraine e-mail: bozhena@litech.lviv.ua

CIN85/Ruk is an adaptor protein involved in such important aspects of cell functioning as ligand-induced endocytosis of receptor tyrosine kinases, regulation of apoptosis and survival, reorganization of actin cytoskeleton and HSV-1 infec­tion. Present work demonstrates that in human lung adenocarcinoma A549 cells CIN85/Ruk is represented by several forms with apparent molecular weights of 130, 100, 85, 70, 60, 56 and 40 kDa. Analysis of subcellular distribution of CIN85/Ruk molecular forms between cytoplasmic and nuclear fractions showed that each form is preferentially localized in certain cellular compartments that may reflect specific roles of CIN85/Ruk molecular forms in cell physiology.

Key words: adaptor proteins, CIN85/Ruk, multiple molecular forms, subcellular distribution.

Adaptor proteins are non-catalytic polypeptides that contain one or more domains and/or motifs able to bind simultaneously to other proteins or non-protein ligands. In such protein clus­ters increased local concentrations of proteins enable low-efficiency interactions, transient bind­ing in a simultaneous or sequential manner resulted in dynamic changes in protein composition that, in turn, provide possibility of prompt and adequate cell reaction to environmental signals that regulate cell growth, proliferation, differentiation, migration and survival [9].

CIN85/Ruk is an adaptor protein structurally composed of three SH3 domains, four blocks of proline-reach sequences and C-terminal coiled-coil region. SH3 domains mediate interaction of CIN85/Ruk with proline-rich motifs-containing proteins [6,7]. And vice versa, the proline-rich region of CIN85/Ruk is crucial for binding to SH3 domain-containing molecules [6], while the coiled-coil region is involved in homo- and heterooligomerization [2,6]. Due to its complex domain organization CIN85/Ruk is functionally implicated in a remarkably wide spectrum of intracellular signalling events involved in down-regulation of receptor tyrosine kinases through induction of their endocytosis [2,10], regulation of apoptosis and cell survival [4], reorganization of cell cytoskeleton [5] and HSV-1 infection [8].

Until now, several CIN85/Ruk molecular forms have been identified [1,2]. It was shown that they are products of alternative splicing and different promoter usage [1]. The appearance of additional molecular forms may be a result of post-translational modification of protein prod­ucts through phosphorylation, ubiquitination and limited proteolysis, caused by the presence of PEST-motifs in C-terminal half of polypeptide chain [11]. CIN85/Ruk molecular forms differ in the domains that they comprise in their structure. As each domain has distinct binding prefer­ences, different CIN85/Ruk forms could be predicted to have significantly divergent binding partners and roles in cell functioning [3].

Knowledge of CIN85/Ruk molecular forms subcellular localization is absolutely neces­sary for a deeper appreciation of how this complex multidomain adaptor protein functions. So,

© Vynnytska B., Bobak Ya., Basaraba O. et al., 2007

the aim of the present work was to study the expression level and subcellular distribution of CIN85/Ruk forms in human lung adenocarcinoma cell line A549.

Human lung adenocarcinoma A549 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/ml penicillin, 50 ug/ml streptomycin in humidified atmosphere containing 5% CO2 at 37°C. To obtain total cell lysates, cells were lysed in 50 mM Tris-HCl, pH 6.8, 2% SDS. To obtain deter­gent-soluble and detergent-insoluble cellular fractions, cells were lysed in extraction buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 5 mM benzamidine, 1 mM PMSF, 10 ug/ml aprotinin, 10 ug/ml leupeptin, 1 ug/ml pepstatin) for 20 min at 4°C. Triton X-100-soluble and Triton X-100-insoluble fractions were resolved by centrifugation at 12000 rpm for 30 min at 4°C. The pellet (detergent-insoluble cel­lular fraction) was dissolved in 50 mM Tris-HCl, pH 6.8, 2% SDS.

To obtain cytosolic and nuclear subcellular fractions, cells were lysed in hypotonic buffer (10 mM HEPES, pH 7.9, 0.6% NP-40, 1.5 mM MgCl2, 10 mM KCl, 1 mM Na3VO4, 5 mM benzamidine, 1 mM PMSF, 10 ug/ml aprotinin, 10 ug/ml leupeptin, 1 ug/ml pepstatin). After centrifugation at 3000 rpm for 15 min at 4°C the supernatant (crude cytosolic fraction) and nuclear pellets were resolved and further lysed separately in extraction buffer to generate Triton X-100-soluble cytosolic fraction (proteins of cytosol and cell membranes), Triton X-100-soluble nuclear fraction (proteins of nucleoplasm and nuclear membranes) and Triton X-100-insoluble nuclear fraction (nuclear matrix- and chromatin-assosiated proteins). Proteins were analyzed by SDS-PAGE 5-18 %, followed by Western blot analysis with rabbit polyclonal anti-Ruks antibodies recognizing common for all CIN85/Ruk forms C-terminal coiled-coil region. Secondary horseradish peroxidase-conjugated anti-rabbit IgG antibodies and ECL (enhanced chemiluminescence) system were used for detection of immunoreactive proteins.

Multiple immunoreactive bands that correspond to proteins with apparent molecular weights of 130, 85, 70, 56 and 40 kDa were revealed in A549 total cell lysate. According to the current experimental data these multiple molecular forms of CIN85/Ruk could be the result of alternative splicing of CIN85/Ruk pre-mRNA transcript (full-length form - 85 kDa, form with­out first SH3A domain - 70 kDa and form without two SH3 domains - 56 kDa), possible post-translational modification through ubiquitination and limited proteolysis [11] (130 and 40 kDa forms respectively) (fig. 1,1).

Analysis of distribution of CIN85/Ruk forms between Triton X-100-soluble and Triton X-100-insoluble cellular fractions showed that forms of 130, 85 and 56 kDa are mainly local­ized in detergent-soluble fraction that argues in favor of their preferential distribution in cell cytoplasmic compartment. In contrast, forms with apparent molecular weights of 70 and 40 kDa were detected both in Triton X-100-soluble and Triton X-100-insoluble cellular fractions, which suggests that these forms could be also associated with cell cytoskeleton and/or nuclear matrix. The form of 60 kDa, which was not present in the total cell lysate, was detected in Triton X-100 -insoluble cellular fraction. The obtained data show that the expression level of 60 kDa form in A549 cells is low and this form is strongly associated with proteins of cell cytoskeleton and/or nuclear matrix (fig. 1,23).

As these data demonstrate specific CIN85/Ruk molecular forms allocation between cel­lular detergent-soluble and detergent-insoluble fractions, further the more precise analysis of CIN85/Ruk forms distribution between A549 cytosolic and nuclear subcellular fractions was performed (fig. 2).

It was shown that the expression level of CIN85/Ruk full-length form (85 kDa) is low and that it is mainly localized in the Triton X-100-soluble cytosolic fraction. The form of

Fig. 2. Subcellular distribution of CIN85/Ruk multiple molecular forms in A549 cells. 1 - Triton X-100-soluble cytosolic fraction; 2 - Triton X-100-soluble nuclear fraction; 3 - Triton X-100-insoluble nuclear fraction; IB - immunoblotting.

130 kDa was detected in Triton X-100-soluble cytosolic fraction and was completely absent in the nuclear fractions. These results argue in favor of preferential distribution of 85 and 130 kDa CIN85/Ruk forms in the cell cytoplasmic compartment. In contrast, the forms of 70 and 40 kDa were localized in nuclear fractions while were barely detected in Triton X-100-soluble cytosolic fraction. These forms were equally distributed between detergent-soluble and detergent-insoluble nuclear fractions. The obtained data suggest possible localization of 70 and 40 kDa CIN85/Ruk molecular forms in nucleoplasm as well as their association with proteins of nuclear matrix and chromatin. The CIN85/Ruk form with apparent molecular weight of 56 kDa was mainly localized in Triton X-100-soluble cytosolic and nuclear fractions and was completely absent in detergent-insoluble nuclear fraction. This argues in favor of preferential distribution of the CIN85/Ruk form of 56 kDa in the cytoplasmic compartment and nucleoplasm. In Triton X-100-insoluble nuclear fraction the form of 100 kDa, which was not present in the total cell lys-ate, was detected, suggesting that this form is expressed in A549 cells at a very low level and is strongly associated with proteins of nuclear matrix and chromatin (fig. 2).

Thus, each subcellular compartment of A549 cells is characterized by specific set of CIN85/Ruk molecular forms. The forms of 100, 70, 60 and 40 kDa are present in fraction of nuclear matrix- and chromatin-associated proteins. In fraction of nucleoplasm and nuclear membranes the CIN85/Ruk forms of 70, 56 and 40 kDa are localized. At the same time cyto-plasmic compartment is characterized by the presence of CIN85/Ruk forms of 130, 85 and 56 kDa. Differential distribution of CIN85/Ruk multiple molecular forms between A549 subcel-lular fractions could be connected with specific and diverse signaling functions that these forms exert in each cellular compartment.

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2. Dikic I. CIN85/CMS family of adaptor molecules // FEBS Letters. 2002. Vol. 529.

P. 110-115.

3. Finniss S., Movsisyan A., Billecke Ch. et al. Studying protein isoforms of the adaptor SETA/CIN85/Ruk with monoclonal antibodies // Biochem. Biophys. Res. Commun. 2004. Vol. 325. N 1. P.174-182.

4. Gout I., Middleton G., Adu J. et al. Negative regulation of PI-3 kinase by Ruk, a novel

adaptor protein // EMBO J. 2000. Vol. 19. P. 4015-4025.

5. Hutchings N. J., Clarkson N., Chalkley R. et al. Linking the T cell surface protein CD2 to

the actin-capping protein CAPZ via CMS and CIN85 // JBC. 2003. Vol. 278. P. 22396­22403.

6. Kowanets K., Husnjak K., Holler D. et al. CIN85 associates with multiple effectors con­trolling intracellular trafficking of epidermal growth factor receptors // Mol. Biol. Cell. 2004. Vol. 15. P. 3155-3166.

7. Kowanetz K., Szymkiewicz I., Haglund K. et al. Identification of a novel proline-arginine motif involved in CIN85-dependent clustering of Cbl and down-regulation of epidermal growth factor receptors // J. Biol. Chem. 2003. Vol. 278. N 41. P. 39735-39746.

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B. Vynnytska, Ya. Bobak, O. Basaraba et al.

10. Soubeyran Ph., Kowanets K., Szymkewicz I. et al. Cbl-CIN85-endophilin complex me­diates ligand-induced downregulation of EGF receptor // Nature. 2002. Vol. 416.

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РОЗПОДІЛ МНОЖИННИХ МОЛЕКУЛЯРНИХ ФОРМ АДАПТЕРНОГО БІЛКА CIN85/RUK МІЖ ЦИТОЗОЛЬНОЮ ТА ЯДЕРНОЮ ФРАКЦІЯМИ КЛІТИН А549 АДЕНОКАРЦИНОМИ ЛЕГЕНІ ЛЮДИНИ

Б. Винницька**, Я. Бобак*, О. Басараба*, Н. Ігуменцева*, Л. Дробот*

* Інститут біології клітини НАН України вул. Драгоманова 14/16, Львів 79005, Україна ** Львівський національний університет імені Івана Франка вул. Грушевського, 4, Львів, 79005, Україна e-mail: bozhena@litech.lviv.ua

Адаптерний білок CIN85/Ruk залучений до формування надмолеку­лярних комплексів, що беруть участь у регуляції важливих для життєдіяль­ності клітини процесів, зокрема, негативній регуляції рецепторних тирози-нових кіназ, реорганізації актинового цитоскелета, регуляції апоптозу та виживання клітин, інфікуванні клітин HSV-1. У клітинах аденокарциноми легень людини лінії А549 CIN85/Ruk представлений формами з молекуля­рними масами 130, 100, 85, 70, 60, 56 та 40 кДа. Аналіз розподілу множин­них молекулярних форм CIN85/Ruk між цитоплазматичними та ядерними субклітинними фракціями засвідчив, що кожна форма CIN85/Ruk локалізо­вана переважно в певному клітинному компартменті. Особливості субклі­тинного розподілу множинних молекулярних форм адаптерного білка CIN85/Ruk можуть відображати специфіку їхньої фізіологічної ролі в фун­кціонуванні клітин.

Ключові слова: адаптерний білок, CIN85/Ruk, множинні молекулярні фор­ми, субклітинні фракції.

Стаття надійшла до редколегії 20.05.06 Прийнята до друку 16.06.06

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B Vynnytska, Ya Bobak, O Basaraba - Distribution of adaptor protein cin85ruk multiple molecular forms between cytosolic and nuclear fractions in human lung adenocarcinoma cell line