Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

Abstract

We previously proposed a model of Class IA PI3K regulation in which p85 inhibition of p110␣ requires (i) an inhibitory contact between the p85 nSH2 domain and the p110␣ helical domain, and (ii) a contact between the p85 nSH2 and iSH2 domains that orients the nSH2 so as to inhibit p110␣. 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 ( c Ϸ12.7 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 p110␣ C2 domain (residue N345) and the p85 iSH2 domain (residues D560 and N564). p85ni-D560K/N564K shows reduced inhibition of p110␣, similar to the truncated p85ni-572 STOP . Conversely, wild-type p85ni poorly inhibits p110␣N345K. Strikingly, the p110␣N345K mutant is inhibited to the same extent by the wild-type or truncated p85ni, suggesting that mutation of p110␣-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.cancer ͉ glioblastoma ͉ phosphoinositide 3-kinase ͉ PIK3CA P I 3-kinases are important cellular regulators of growth, survival, and motility, and deregulation of PI 3-kinase signaling contributes to cancer and other human diseases (1). Class IA PI 3-kinases, which produce PI[3,4,5]P3 in intact cells (2), are obligate heterodimers of a regulatory subunit (p85␣, p85␤, p55␣, p50␣, or p55␥) and a catalytic subunit (p110␣, p110␤, or p110␦) (reviewed in ref.3). The regulatory subunits have two major functions: they stabilize the catalytic subunits against thermal denaturation, and they maintain the catalytic subunit in an inhibited, low activity state (4, 5).p85 and p110 are both multidomain proteins that bind to each other and to upstream activators such as Rac and Cdc42, Ras, and tyrosine phosphorylated receptors and adapters (reviewed in ref. 6). p85 contains an SH3 domain, a Rac/Cdc42-binding domain homologous to a GAP domain in the BCR gene product, and two SH2 domains that flank an antiparallel coiled coil domain (the iSH2 domain). While NMR, EPR, and crystal structures have been obtained for the individual domains (7-15), there are currently no structures that define how these domains are arranged in space. The p110␣ catalytic subunit has been better defined, with structures of the N-terminal adapter-binding domain (ABD) or the entire p110␣ bound to the coiled coil (iSH2) domain of p85 (15,16). Like the related Class IB catalytic subunit p110␥ (17), p110␣ contains Ras-binding, C2, ...

Cancer-derived mutations in the regulatory subunit p85α of phosphoinositide 3-kinase function through the catalytic subunit p110α

“…Expression of mutant or WT exogenous p85 in CEF also induces elevated levels of p110α by stabilizing the catalytic subunit (17,29). The observations are in agreement with published studies on p85 mutants that used different cellular systems (17,19). Going beyond these previous studies, we demonstrate oncogenic activity in a comprehensive collection of mutated p85, derived from glioblastoma.…”

“…The results described in this communication are in agreement with the hypothesis that the gain-of-function mutations in p85 destabilize the inhibitory interaction between p85 and p110, resulting in a relief of p110 inhibition (17)(18)(19). At the same time, these mutants retain the ability to bind to p110, probably by the interaction with the adapter-binding domain, thus stabilizing p110.…”

“…These mutations show oncogenic potency in cell culture and elevated levels of downstream signaling and operate through the p110α isoform of the catalytic subunit of class I PI3K. Our observations extend recent studies of the p85α mutants using different cell systems (17,19) by providing quantitative data on the oncogenic potency of the mutations and by presenting evidence that suggests a unique role of p110α for the p85 mutation-induced gain of function in PI3K activity. …”

“…Breaking the α helix would disturb the positioning of the nSH2 and cSH2 (C-terminal SH2) domains. In addition, both of these mutations are in close proximity to the C2 domain of p110α, and disruption of the α helix could disrupt interactions with the C2 domain, further increasing catalytic activity as shown in a recent study (19). These two mutations along with the C420R mutation of p110α probably represent one of the mechanisms for aberrant activation of PI3K.…”

“…The mutations cluster in the inter-SH2 (iSH2) domain of p85α, involving residues that interact with the C2 domain of the catalytic subunit p110α (15,17). The iSH2-C2 domain interaction has an inhibitory effect on enzyme activity, and the mutations in the iSH2 domain of p85α could weaken this interaction and release the inhibition of PI3K activity (15,(17)(18)(19). A similar mechanism has been proposed for the gain-of-function mutations in the helical domain of p110α that alleviate an inhibitory interaction with the N-terminal SH2 domain (nSH2) of p85α (20).…”