Complexes of g-tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells Vitaliy Kukharskyy, Vadym Sulimenko, Libor Macu ˚rek, Tetyana Sulimenko, Eduarda Dra ´berova ´, and Pavel Dra ´ber * Institute of Molecular Genetics, Department of Biology of Cytoskeleton, Academy of Sciences of the Czech Republic, Prague 4, Czech Republic Received 2 October 2003, revised version received 8 April 2004 Available online 10 May 2004 Abstract Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that g-tubulin (g-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, g-tubulin, and with anti-phosphotyrosine antibody revealed that g-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in g-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated g-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing g-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of g-tubulin interaction with tubulin dimers or other proteins during neurogenesis. D 2004 Elsevier Inc. All rights reserved. Keywords: Antibodies; Fyn kinase; Gamma-tubulin; Neuronal differentiation; P19 cells; Src kinase Introduction It is well established that the development of nervous system is regulated by a variety of protein tyrosine kinases. While the neural functions of receptor tyrosine kinases are sufficiently characterized, the functions of nonreceptor ty- rosine kinases, including kinases pp60 src (Src) and p59 fyn (Fyn) that belong to the family of Src kinases, are not fully understood. Src family kinases have been implicated in events regulating neuronal differentiation and function of neuronal cells [1–3]. The kinases could modulate microtu- bule dynamics during neurite outgrowth as a fraction of tubulin associated with plasma membrane serves as a substrate for Src kinase [4,5]. Both Src and Fyn kinases were found in complexes containing cell surface receptors and tubulin [6]. Embryonal carcinoma cell line P19 is a suitable model system for studying the molecular mechanisms underlying differentiation and early embryonic development. P19s are murine multipotent cells that can differentiate in culture into neural cells when aggregated and subsequently cultured in the presence of a nontoxic concentration of all-trans-retinoic acid (RA) [7]. There are substantial changes in the expres- sion of microtubule proteins during neuronal differentiation of P19 cells [8–10], and both Src and Fyn kinases are present in differentiated cells [11]. 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.04.016 Abbreviations: EC, embryonal carcinoma; Fyn, protein tyrosine kinase p59 fyn ; GST, glutathione S-transferase; MTOC, microtubule organizing center; MSB, microtubule-stabilizing buffer; PBS, phosphate buffered saline; PP2, Src-family selective tyrosine kinase inhibitor; PP3, negative control for inhibitor PP2; P-Tyr, protein phosphorylated on tyrosine; RA, all-trans-retinoic acid; SDS-PAGE, one-dimensional SDS-polyacrylamide gel electrophoresis; 2D-PAGE, two-dimensional electrophoresis; Src, protein tyrosine kinase pp60 src ; SH2 and SH3, Src homology 2 and 3 domains; a-Tb, a-tubulin; Aca-Tb, acetylated a-tubulin; hIII-Tb, h-tubulin class III; g-Tb, g-tubulin; gTuRC, g-tubulin-ring complex; gTuSC, g- tubulin small complex; TBST, 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.05% (v/v) Tween 20. * Corresponding author. Institute of Molecular Genetics, Department of Biology of Cytoskeleton, Academy of Sciences of the Czech Republic, Vı ´den ˇska ´ 1083, 142 20 Prague 4, Czech Republic. Fax: +420-241-062-758. E-mail address: paveldra@biomed.cas.cz (P. Dra ´ber). www.elsevier.com/locate/yexcr Experimental Cell Research 298 (2004) 218 – 228