Spatial cycles mediated by UNC119 solubilisation maintain Src family kinases plasma membrane localisation

Antonios D Konitsiotis,Angel Stanoev,Lisaweta Roßmannek,Malte Schmick,Philippe I H Bastiaens

doi:10.1038/s41467-017-00116-3

Abstract

The peripheral membrane proto-oncogene Src family protein tyrosine kinases relay growth factor signals to the cytoplasm of mammalian cells. We unravel the spatial cycles of solubilisation, trapping on perinuclear membrane compartments and vesicular transport that counter entropic equilibration to endomembranes for maintaining the enrichment and activity of Src family protein tyrosine kinases at the plasma membrane. The solubilising factor UNC119 sequesters myristoylated Src family protein tyrosine kinases from the cytoplasm, enhancing their diffusion to effectively release Src family protein tyrosine kinases on the recycling endosome by localised Arl2/3 activity. Src is then trapped on the recycling endosome via electrostatic interactions, whereas Fyn is quickly released to be kinetically trapped on the Golgi by palmitoyl acyl-transferase activity. Vesicular trafficking from these compartments restores enrichment of the Src family protein tyrosine kinases to the plasma membrane. Interference with these spatial cycles by UNC119 knockdown disrupts Src family protein tyrosine kinase localisation and signalling activity, indicating that UNC119 could be a drug target to affect oncogenic Src family protein tyrosine kinase signalling.

Highlights

The peripheral membrane proto-oncogene Src family protein tyrosine kinases relay growth factor signals to the cytoplasm of mammalian cells
We examined whether these structures corresponded to the Golgi apparatus since Fyn-mCit is dually palmitoylated on two C-terminal cysteines and palmitoylation of N/HRas has been shown to predominantly occur on the Golgi[13]
This was not affected by long-term incubation with the protein synthesis inhibitor, cyclohexamide, demonstrating that the temperature block induced a shift in the steady-state distribution of Fyn-mCit from predominantly plasma membrane (PM) to the Golgi (Supplementary Fig. 2c)

Summary

Introduction

The peripheral membrane proto-oncogene Src family protein tyrosine kinases relay growth factor signals to the cytoplasm of mammalian cells. We unravel the spatial cycles of solubilisation, trapping on perinuclear membrane compartments and vesicular transport that counter entropic equilibration to endomembranes for maintaining the enrichment and activity of Src family protein tyrosine kinases at the plasma membrane. Src is trapped on the recycling endosome via electrostatic interactions, whereas Fyn is quickly released to be kinetically trapped on the Golgi by palmitoyl acyl-transferase activity Vesicular trafficking from these compartments restores enrichment of the Src family protein tyrosine kinases to the plasma membrane. A major class of peripheral membrane kinases is the lipidated Src family protein tyrosine kinases (SFKs) that amplify and sustain signalling from activated receptors[2,3,4], which explains their oncogenic potential[4, 5]. The spatial organisation of the Arl-mediated release of SFKs from UNC119 is analogous to that of Ras protein release from PDEδ, revealing a conserved mechanism to maintain the localisation of lipidated peripheral membrane molecules

Methods

Results

Conclusion