Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110alpha and are disrupted in oncogenic p85 mutants.
We previously proposed a model of Class IA PI3K regulation in which p85 inhibition of p110alpha requires (i) an inhibitory contact between the p85 nSH2 domain and the p110alpha helical domain, and (ii) a contact between the p85 nSH2 and iSH2 domains that orients the nSH2 so as to inhibit p110alpha. We proposed that oncogenic truncations of p85 fail to inhibit p110 due to a loss of the iSH2-nSH2 contact. However, we now find that within the context of a minimal regulatory fragment of p85 (the nSH2-iSH2 fragment, termed p85ni), the nSH2 domain rotates much more freely (tau(c) approximately 12.7 ns) than it could if it were interacting rigidly with the iSH2 domain. These data are not compatible with our previous model. We therefore tested an alternative model in which oncogenic p85 truncations destabilize an interface between the p110alpha C2 domain (residue N345) and the p85 iSH2 domain (residues D560 and N564). p85ni-D560K/N564K shows reduced inhibition of p110alpha, similar to the truncated p85ni-572(STOP). Conversely, wild-type p85ni poorly inhibits p110alphaN345K. Strikingly, the p110alphaN345K mutant is inhibited to the same extent by the wild-type or truncated p85ni, suggesting that mutation of p110alpha-N345 is not additive with the p85ni-572(STOP) mutation. Similarly, the D560K/N564K mutation is not additive with the p85ni-572(STOP) mutant for downstream signaling or cellular transformation. Thus, our data suggests that mutations at the C2-iSH2 domain contact and truncations of the iSH2 domain, which are found in human tumors, both act by disrupting the C2-iSH2 domain interface.
Wu
Haiyan
H
Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Shekar
S Chandra
SC
Flinn
Rory J
RJ
El-Sibai
Mirvat
M
Jaiswal
Bijay S
BS
Sen
K Ilker
KI
Janakiraman
Vasantharajan
V
Seshagiri
Somasekar
S
Gerfen
Gary J
GJ
Girvin
Mark E
ME
Backer
Jonathan M
JM
D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006367:HeLa Cells; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*
D019869:Phosphatidylinositol 3-Kinases