Assembly of novel, nuclear dimers of the PI3-Kinase regulatory subunits underpins the pro-proliferative activity of the Cdc42-activated tyrosine kinase, ACK

Abstract

The tyrosine kinase ACK is an oncogene associated with poor prognosis in human cancers. ACK promotes proliferation, in part, by contributing to the activation of Akt, the major PI3-Kinase effector. We show that ACK also regulates PI3-Kinase directly, via interactions with the PI3-Kinase regulatory subunits. ACK interacts with all five regulatory subunit isoforms and directly phosphorylates p85α, p85β, p55α and p50α on Tyr607 (or equivalent). Phosphorylation of p85β at this residue promotes cell proliferation but, counterintuitively, ACK does not stimulate PI3-Kinase catalytic activity. We show that ACK stabilizes p85α levels by promoting an interaction between the p85 nSH2 domain and pTyr607, protecting p85 from ubiquitination. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions where the increased levels of the regulatory subunits, together with the nSH2-pTyr607 interaction, promote formation of dimeric p85. We postulate that these novel dimers undertake nuclear functions that contribute to Cdc42-ACK driven oncogenesis. We propose that ACK shapes PI3-Kinase signalling by dampening the PIP 3 response, whilst continuing to drive cell proliferation through Akt activation and hereto unexplored but crucial functions of nuclear dimeric p85. These new regulatory subunit dimers represent a previously undescribed mode of regulation for PI3-Kinase and potentially reveal additional avenues for therapeutic intervention.

Class IA PI3K regulatory subunits: p110-independent roles and structures

“…The site of this phosphorylation was not identified but could, of course, be Tyr607 on p85α, the target site for ACK, which is activated upon insulin stimulation [54]. This would fit with our recent findings of nuclear localised C-terminal dimers of p85 [41].…”

“…C-terminal dimers are instigated by phosphorylation on a conserved tyrosine residue in the iSH2 region; Tyr607 for p85α. p85α is phosphorylated downstream of the insulin receptor at Tyr607 [40] and we have found that it is directly phosphorylated by the non-RTK ACK [41]. ACK binds to all five isoforms of the regulatory subunits and phosphorylates four of them.…”

“…C-terminal mediated dimers of the regulatory subunits are stabilised and protected from UPS-mediated degradation, as ACK protects wt p85α from degradation but not a non-phosphorylatable mutant Y607F p85α. In addition, ACK completely abolished polyubiquitination of p85α [41]. p42, the short isoform of ErbB3-binding protein 1 (Ebp1) is a tumour suppressor protein that targets the iSH2 region of p85α, bringing it into close proximity to its E3 ligase (the HSP70/CHIP complex) [42].…”

“…C-terminal dimers of p50α/ p85β were predominantly found in nuclear-enriched cell fractions compared with N-terminal dimers of p85α/ p85β, which were cytoplasmic. Complexes of ACK and the PI3K regulatory subunits were also found in the nuclear-enriched fractions, despite the individual components showing dual localisation, and pTyr607 p85α was also predominantly nuclear [41]. The C-terminal dimers are regulated by phosphorylation and only form under conditions where ACK is active (the regulatory mechanisms and signalling pathways ACK controls have been recently reviewed [43]).…”

“…The C-terminal dimers are regulated by phosphorylation and only form under conditions where ACK is active (the regulatory mechanisms and signalling pathways ACK controls have been recently reviewed [43]). Experiments using stable cell lines expressing mutant versions of p85 showed phosphorylation of p85β drives cell proliferation [41]. The functions of these C-terminal p85 dimers remain to be determined but it is likely that they have important pro-proliferative roles in the nucleus.…”