Class IA phosphoinositide 3-kinases are obligate p85-p110 heterodimers

Abstract

Class IA phosphoinositide 3-kinases (PI3Ks) signal downstream of tyrosine kinases and Ras and control a wide variety of biological responses. In mammals, these heterodimeric PI3Ks consist of a p110 catalytic subunit (p110␣, p110␤, or p110␦) bound to any of five distinct regulatory subunits (p85␣, p85␤, p55␥, p55␣, and p50␣, collectively referred to as ''p85s''). The relative expression levels of p85 and p110 have been invoked to explain key features of PI3K signaling. For example, free (i.e., non-p110-bound) p85␣ has been proposed to negatively regulate PI3K signaling by competition with p85/p110 for recruitment to phosphotyrosine docking sites. Using affinity and ion exchange chromatography and quantitative mass spectrometry, we demonstrate that the p85 and p110 subunits are present in equimolar amounts in mammalian cell lines and tissues. No evidence for free p85 or p110 subunits could be obtained. Cell lines contain 10,000 -15,000 p85/p110 complexes per cell, with p110␤ and p110␦ being the most prevalent catalytic subunits in nonleukocytes and leukocytes, respectively. These results argue against a role of free p85 in PI3K signaling and provide insights into the nonredundant functions of the different class IA PI3K isoforms.quantitative mass spectrometry ͉ signaling ͉ protein stability ͉ gene knockout ͉ lipid kinase P hosphoinositide 3-kinases (PI3Ks) generate lipid second messengers that serve as membrane docking sites for a variety of downstream effector proteins such as protein kinases, regulators of small GTPases, and scaffolding proteins (1, 2). The class IA PI3Ks are heterodimers consisting of a p110 catalytic subunit and a smaller regulatory subunit with Src-homology 2 (SH2) domains. Mammals have three catalytic subunits (p110␣, p110␤, p110␦) and five regulatory subunits (p85␣, p85␤, p55␥, p55␣, p50␣) (1, 2). Under experimental conditions, each p110 isoform can bind any p85 isoform with no apparent preference (see among others, refs. 3 and 4). p85s have a dual effect on the p110 subunits because they stabilize the thermally labile p110s but also conformationally inhibit their catalytic activity (5). Upon cellular stimulation, SH2 domain-mediated recruitment of p85/p110 complexes to Tyr phosphorylated (pY) membraneproximal proteins serves dual functions: It positions p110 in proximity with its substrates, and the engagement of the p85 SH2 domains relieves p85-mediated inhibition of p110, thus increasing enzymatic activity of p110 (6, 7).Experiments using gene-targeted mice and p110 isoformselective inhibitors have uncovered nonredundant physiological functions of the p110 isoforms. These functions include insulin signaling in metabolic tissues (p110␣; refs. 8 and 9), integrin signaling in platelets (p110␤; ref. 10) and signaling through a variety of receptors in leukocytes (p110␦; refs. 11-15). The mechanisms of this nonredundant signaling are not fully understood. Indeed, p110 isoforms have high homology in their primary sequence, interact nonselectively with the different p85s, and have the same lipid...

Neuregulin 1-ErbB4-PI3K signaling in schizophrenia and phosphoinositide 3-kinase-p110δ inhibition as a potential therapeutic strategy

“…Interestingly, PIK3CD associates in vitro, nonselectively with p85 regulatory subunits (25) and targeted deletion of the PIK3CD gene in mice produces compensatory changes in the p85 and p55 regulatory genes (32). Thus, we hypothesized that the change in PIK3R3 was a secondary effect, i.e., that expression of PIK3CD would predict expression of PIK3R3 and account for its relative increase.…”

“…Unlike ErbB4 CYT-2, ErbB4 CYT-1 includes a phosphoinositide 3-kinase (PI3K)-binding site and is capable of activating the PI3K pathway (21)(22)(23)(24). Class IA PI3Ks are activated by receptor tyrosine kinases and exist as obligate heterodimers (25,26) consisting of a p110 catalytic subunit (PIK3CA, PIK3CB, and PIK3CD encode the isoforms p110α, p110β, and p110δ, respectively) and a p85 regulatory subunit (PIK3R1, PIK3R2, and PIK3R3, which encode the p85α, p85β, and p55γ isoforms, respectively) (25). PI3K signaling in turn mediates NRG1-induced cell survival and neuronal and synaptic development (21)(22)(23)(24).…”

p85β phosphoinositide 3-kinase subunit regulates tumor progression

“…p85β is expressed at lower levels than p85α in most tissues (5)(6)(7). Whereas mice deficient in Pik3r2 develop normally and exhibit only moderate metabolic and immunological defects (7) Pik3r1 −/− mice die perinatally (8).…”

Calpain interacts with class IA phosphoinositide 3-kinases regulating their stability and signaling activity

“…Accordingly, several mechanisms to control PI3K activity have evolved, some of which (highlighted in the introduction) are relatively well characterized (9,10,15,19,21,37). Regulation by protein-protein interactions is a recurrent theme in biochemistry (12,13) and several signaling pathway components are known to be regulated by the formation of protein complexes (14)(15)(16)(17).…”

“…It is known that binding of p85 to p110 leads to a decrease in the lipid kinase activity of p110 in vitro (18). However, whether p85 regulates p110 activity dynamically in vivo is not clear as this interaction is very strong and both subunits are constitutively bound in cells (19). Ras binding to the p110α subunit was deduced by structural analysis and, although this interaction is known to be critical for some PI3K functions (20,21), it does not survive biochemical purification, indicating that it is a weak and transient interaction.…”