ment was identical to that induced by the H1047R mutant of p110α. The same elevated cellular growth rates were found with the K379E mutant. Mutants R574fs, T576del, and DKRMNS560del induced an intermediate enhancement of cell growth that roughly corresponded to their intermediate efficiency of oncogenic transformation. Overexpression of WT p85 or of empty RCAS LY335979 Zosuquidar vector did not produce a detectable effect on the growth rates of CEF. Author contributions: P.K.V. designed research; M.S., P.H., B.T.H., and J.R.H. performed research; M.S., P.H., B.T.H., and J.R.H. contributed new reagents/analytic tools; M.S., P.H., B.T.H., J.R.H., and P.K.V. analyzed data; and M.S., P.H., B.T.H., J.R.H., and P.K.V. wrote the paper. The authors declare no conflict of interest.1M.S. and P.H. contributed equally to this work.
2Present address: Center for Experimental Medicine, Department of Biochemistry and Molecular Biology I, Cellular PI-103 Signal Transduction, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg, Germany.3To whom correspondence should be addressed. E-mail: pkvogtscripps. This article contains supporting information online at pnas/lookup/suppl/doi:10.1073/pnas.1009652107/-/DCSupplemental. pnas/cgi/doi/10.1073/pnas.1009652107 PNAS | August 31, 2010 | vol.107 | no.35 | 15547�?5552 MEDICAL SCIENCES Mutations in p85 Induce Elevated Levels of Downstream Signaling. As a regulatory subunit of PI3K, p85 signals in conjunction with the catalytic subunit p110 through the phosphorylation of phosphoinositide 4,5-bisphosphate, generating phosphoinositide 3,4,5-trisphosphate.
The trisphosphate recruits the serine-threonine kinase Akt and its activating kinase PDK1. Akt then initiates a cascade of downstream phosphorylations that activate TOR , S6K , and 4E-BP1. We have examined the activating phosphorylation of Akt and of 4E-BP1 as indicators of the PI3K signaling pathway. CEF transfected with the mutant constructs were analyzed by Western blot. Transfection with the H1047R mutant of p110α served as positive control, and transfection with empty RCAS vector or WT p85 was used as negative control. All p85 mutants stimulated phosphorylation of Akt and of 4E-BP1. The strong differences in potency seen in the cell transformation assay were not evident in the levels of Akt or 4E-BP1 phosphorylation. These data support the conclusion that the mutations in p85 induce a gain of enzymatic function of PI3K.
They also suggest, as has been observed previously, that potency in cell transformation is not always correlated with signaling levels measured by the phosphorylation of Akt and other downstream targets. Mutant p85 Proteins Still Bind to the Catalytic Subunits p110α and p110β. The p85 mutations cluster in regions of the protein that interact with the C2 and the helical domains of the catalytic subunit p110α. These interactions mediate an inhibition of the catalytic activity of the enzyme, hence themutant phenotypes could result from a weakening of p85–p110 binding. We therefore determined the ability of the p85mutants to bind to p110α and p110β,the two ubiquitously expressed isoforms of p110. FLAG-tagged p85 constructs were coexpressed with human p110α or p110β in 293T cells using the pCAGGSvector.
Coimmunoprecipitation and Western blots of cell lysates were performed as outlined in the legend to Fig.5. All p85 mutants retained the ability to interact with the p110α and p110β isoforms of the catalytic subunit, and there was no significant reduction in this binding activity. Phenotypic Effects of p85 Mutations Are Mediated Exclusively by p110α. The ability of the p85 mutants to bind to p110α as well as p110β raises the question of which catalytic isoform is t