JBC 2005: Src phosphorylation of Alix (By Bogler group)

Title: Src phosphorylation of Alix/AIP1 Modulates Its Interaction with Binding Partners and Antagonizes Its Activity

Authors: Schmidt MHH and Bogler O. They published several papers on Alix.

Main points:
1) Alix Y319 is required for Src SH2 and FL binding; P745P752P755 is the sites for Src SH3 binding (close to SETA/CIN85 binding site, but not overlapped); and Y319 itself is sufficient for the binding.

2) Src phosphorylates and binds Y319. After that, Src further hyperphosphorylates Alix PRD (p3419). However, the hyperphosphorylation isn't the requirement of Alix/Src interaction.

3) Src's influence to Alix localization (p3419): Src/Alix binding relocates p95 from cytoskeleton to cytoplasm. (Fig.4)

4) Src/Alix to CIN85 (reduction effect): Fig 5 A & B
When co-transfections of Src, Alix and CIN85 to cells (HeLa), CIN85 reduction can be clearly observed. This reduction can be inhibited by proteasomal/lysosomal inhibitor, or by deleting aa 784-869 of Alix (Alix PRD). Means what?
The authors said " the reduction in CIN85 is a consequence of Alix and Src activity ... ".
Here is my guess: Src/Alix relocates Alix into cytoplasm --> cytoplasmic Alix works in MVB pathway --> more proteins are sorted into late endosome and lysosome --> more CIN85 degradation occurs.
And, p95 delta784-869 cannot be hyperphosphorylated, so it may not be able to dissociate with cytoskeleton or can't work in MVB (purely my guesses).

5) Alix/Src interaction to RTK turnover
Alix inhibits RTKs internalization
Src neuralizes Alix's inhibition. The neuralization effects depend (partially) on Alix PRD (Fig 7A, ln 4).
More interestingly, Y319F Alix is similar with mock to RTK internalization. Src cannot interact with it --> so Src cannot inhibit Alix.
But Y319F totally has no effect --> it loses functions in this aspect --> Y319 is very crucial for Alix's function in RTK internalization, and may be in other aspects.

Suggestions to us: maybe I'd try to make a Y319F Hp95 in retroviral vectors and exam its effects to cell adhesion, actin stress fiber assembly and FN assembly. It is quite possible that this mutant p95 can't work well in some of these aspects. Then further works can be done in this direction. And the best thing is that we have the specific mAb for this site! :D