Elsevier

Experimental Cell Research

Volume 298, Issue 1, 1 August 2004, Pages 218-228
Experimental Cell Research

Complexes of γ-tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells

https://doi.org/10.1016/j.yexcr.2004.04.016Get rights and content

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 γ-tubulin (γ-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, γ-tubulin, and with anti-phosphotyrosine antibody revealed that γ-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in γ-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated γ-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing γ-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 γ-tubulin interaction with tubulin dimers or other proteins during neurogenesis.

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 tyrosine kinases, including kinases pp60src (Src) and p59fyn (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], [2], [3]. The kinases could modulate microtubule 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 expression of microtubule proteins during neuronal differentiation of P19 cells [8], [9], [10], and both Src and Fyn kinases are present in differentiated cells [11].

Microtubules, known to play an essential role during neurite outgrowth, are dynamic polymers assembled from α- and β-tubulin heterodimers. γ-Tubulin (γ-Tb) [12] is a highly conserved member of the tubulin superfamily that is located on the minus end of microtubules in microtubule organizing centers (MTOCs) [13]. The majority of γ-tubulin is, however, associated with other proteins in soluble cytoplasmic complexes. Large γ-tubulin-ring complexes (γTuRCs) were identified in various species [14], [15]. Besides γTuRC, there also exist smaller complexes (γ-tubulin small complex; γTuSC) [16] that comprise two molecules of γ-tubulin and one molecule each of GCP2 and GCP3 (γ-tubulin complex proteins) [17], [18]. The γ-TuRCs are formed by small complexes and by other proteins. In addition to nucleation from MTOC, γ-TuRCs are also involved in the regulation of the dynamics of microtubule minus ends [19].

γ-Tubulin itself has been recognized as a microtubule minus end binding molecule in not anchored microtubules [20], and the binding sites for α- and β-tubulin subunits on γ-tubulin were identified using synthetic peptides [21]. In brain, soluble γ-tubulin associated with αβ-tubulin dimers irrespective of the size of γ-tubulin complexes [22]. Membrane-bound forms of γ-tubulin with nucleation activity were also described [23], [24].

While it is well known that the αβ-tubulin heterodimers are subject to a large number of posttranslational modifications that are essential for regulation of their functions [25], knowledge about posttranslational modifications of γ-tubulin and its interaction partners is limited. Phosphorylation of γ-tubulin on tyrosine has so far been reported in budding yeast [26], and we found γ-tubulin in rat basophilic leukemia cells in complexes containing protein tyrosine kinase p53/p56lyn belonging to the family of Src kinases [27]. We therefore wanted to find out whether γ-tubulin also interacts with other Src family kinases that are expressed during neuronal differentiation. During this process, substantial changes in cell morphology and microtubule arrangement are known to occur.

Here we report for the first time that γ-tubulin in differentiating P19 cells is phosphorylated and forms complexes with nonreceptor protein tyrosine kinases Src and Fyn. Consequently, the Src family kinases might have an important role in the regulation of γ-tubulin interaction with tubulin dimers or other proteins during neuronal differentiation.

Section snippets

Materials

Immobilized Protein A Plus, Immobilized Protein L Plus, and SuperSignal WestPico Chemiluminescent reagents were bought from Pierce (Rockford, IL, USA) and SYPRO Ruby Protein Gel Stain from Molecular Probes (Leiden, The Netherlands). Protease-inhibitor cocktail tablets (“Complete EDTA-free”) were from Roche Molecular Biochemicals (Mannheim, Germany), and bovine serum albumin was from Serva Feinbiochemica (Heidelberg, Germany). All-trans-retinoic acid (RA), alkaline phosphatase from Escherichia

Expression of kinases and tubulins in differentiating P19 cells

To compare the expression of protein tyrosine kinases of Src family and tubulins during neuronal differentiation of P19 cells, blots of whole cell extracts from unstimulated cells and cells stimulated with RA for 1–12 days were probed with antibodies against kinases Src and Fyn and with antibodies against tubulins. Neuron-specific class III β-tubulin was used as a marker of neuronal differentiation and acetylated α-tubulin as a marker of stable microtubules in newly formed neuronal projections.

Discussion

Quantitative immunoblotting of P19 cells stimulated with retinoic acid revealed that the expression of Src family kinases Src and Fyn was substantially increased during neuronal differentiation documented by typical changes in cell morphology. Elevated expression of kinases corresponded with the increased level of proteins phosphorylated on tyrosine. Although the level of γ-tubulin did not vary during differentiation, substantial changes were observed in its subcellular localization.

Acknowledgements

We thank Dr. H.E. Varmus (Memorial Sloan-Kettering Cancer Center, New York, USA) and Dr. V. Sovová (Institute of Molecular Genetics, Prague, Czech Republic) for providing constructs encoding GST-tagged fusion proteins containing SH2 and SH3 domains of Src. We thank Dr. Petr Dráber (Institute of Molecular Genetics, Prague, Czech Republic) for antibody against GST. This work was supported in part by grants from the Grant Agency of the Czech Academy of Sciences (A5052004), Grant Agency of the

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