Abstract
Src-family kinases, expressed in a wide variety of cell types, are anchored to cellular membranes through posttranslational lipid modifications and involved in diverse cellular signalling. In epithelial cells, Src-family kinases are localized at the plasma membrane and participate in epithelial functions. Epithelial cell polarity is achieved through dynamic reorganization of protein trafficking. To examine the trafficking of Src-family kinases between polarized and non-polarized epithelial cells, we generated an MDCK cell line that can inducibly express a protein of interest in a polarized state at any time. We show here that Lyn, a member of Src-family kinases, mainly localizes to the plasma membrane in polarized MDCK cells and to endomembranes in non-polarized MDCK cells. Cell-cell interactions between adjacent MDCK cells recruit Lyn from endomembranes to the plasma membrane even without cell attachment to extracellular matrix scaffolds, and loss of cell-cell interactions by calcium deprivation relocates Lyn from the plasma membrane to endomembranes through Rab11-mediated recycling. Therefore, using our MDCK cells expressing inducible Lyn, we reveal that calcium-dependent cell-cell interactions play a critical role in plasma membrane localization of Lyn in polarized MDCK cells.
Highlights
Src-family non-receptor tyrosine kinases comprise at least eight members: c-Src, Lyn, c-Yes, Fyn, c-Fgr, Hck, Lck, and Blk
Western blotting analysis showed that treatment of Madin-Darby canine kidney (MDCK)/ tetracycline repressor (TR)/Lyn cells with doxycycline (Dox), a tetracycline derivative, induces expression of human Lyn, whose expression is repressed before Dox treatment (Fig. 1)
We show that calcium-dependent cell-cell interactions dynamically promote plasma membrane-localization of Lyn, a mono-myristoylated and mono-palmitoylated member of Src-family kinases, in polarized MDCK cells
Summary
Src-family non-receptor tyrosine kinases comprise at least eight members: c-Src, Lyn, c-Yes, Fyn, c-Fgr, Hck, Lck, and Blk. Src-family kinases are anchored to the cytoplasmic side of cellular membranes through posttranslational lipid modifications and are involved in transduction of tyrosine phosphorylation signals[2]. A member of Src-family kinases, is expressed in a wide variety of cell types, including epithelial cells, neuronal cells, and hematopoietic cells, and involved in diverse cellular signalling[3,4,5,6]. Src-family kinases, including Lyn, are known to localize predominantly to the plasma membrane in polarized epithelial cells[3], it remains to be elucidated whether establishment of cell polarity affects the trafficking pathway of Src-family kinases. We generated Madin-Darby canine kidney (MDCK) cell lines inducibly expressing Src-family kinases and examined the localization of Lyn in the different culture conditions. The localization of Lyn at the plasma membrane depends on calcium-dependent cell-cell interactions irrespective of cell-scaffold interactions
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