Purchase this article with an account.
Anna Ganios, Shameka J Shelby, Kecia L Feathers, Lin Jia, Debra A Thompson; MERTK Interactions with Src Family Kinases in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2014;55(13):401.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Signaling by the receptor tyrosine kinase MERTK is essential for the phagocytic uptake of shed photoreceptor outer segments by the retinal pigment epithelium (RPE). We have previously shown that MERTK activates the intracellular tyrosine kinase Src, which in turn regulates the Rab effector GDI1. The purpose of the present study is to further define the potential for MERTK activation of Src family kinases (SFKs) during RPE phagocytosis.
The expression of SFKs in the RPE was assessed using RT-PCR, immunohistochemical, and western analysis. MERTK interacting proteins were identified using Ni2+-NTA pulldowns with the intracellular domain of MERTK (aa 571-999) that was expressed as a 6xHis fusion protein, purified using Ni2+-NTA agarose, and phosphorylated by the addition of ATP. 6xHis-rMERTK571-999 was incubated with cDNA clones encoding the SH2-domains of Src, Blk, Fgr, Frk, Fyn, Hck, and Yes that were expressed as GST-fusion proteins and purified using glutathione agarose, or with RPE-J cell homogenates. MERTK-associated proteins present in Ni2+-NTA pulldowns were evaluated on coomassie blue stained gels and by western analysis.
Transcripts encoding multiple SFKs were detected in mouse RPE, with Src, Hck, Fyn, and Yes present at apparently highest abundance. Expression of Src, Fyn, and Yes was confirmed by western analysis of rat RPE/choroid and RPE-J cell homogenates. Immunohistochemical analysis of mouse RPE/retina cryosections showed differential expression patterns of SFKs throughout the layers of the retina. MERTK pulldowns showed interactions with the cloned SH2-domains of Src, Blk, Fgr, Frk, Fyn, Hck, and Yes, and with endogenous Src and Fyn.
We find that signaling downstream of MERTK has the potential to activate multiple SFKs in the RPE. Viewed together with our earlier findings that MERTK activation of Src regulates Rab effectors involved in membrane trafficking, and the known role of Src in regulating cytoskeletal movements, our findings suggest that MERTK activation of SFKs is a central feature of the molecular mechanism of RPE phagocytosis.
This PDF is available to Subscribers Only