Twice upon a time: PI3K’s secret double life exposed Emilio Hirsch, Laura Braccini, Elisa Ciraolo, Fulvio Morello and Alessia Perino Molecular Biotechnology Center, Via Nizza 52, 10126 Torino, Italy Class I phosphoinositide 3-kinases (PI3Ks) are heterodi- meric enzymes involved in signal transduction triggered by growth factors and G-protein-coupled receptors. The catalytic function of PI3Ks is well known to promote a wide variety of biological processes, including prolifer- ation, survival and migration, but a new layer of com- plexity in the function of PI3Ks has recently emerged, indicating that these proteins function not only as kinases but also as scaffold proteins. Knockout mice that lack PI3K protein expression show a different phe- notype from knock-in mice expressing PI3K mutants that have lost their kinase activity, providing evidence for this novel role of PI3Ks. We will discuss such findings, high- lighting the crucial scaffold function of PI3Kg in cAMP homeostasis and PI3Kb in receptor recycling. Setting the stage The enzymatic activity of phosphoinositide 3-kinases (PI3Ks) is essential in eukaryotic cells to regulate many processes, such as cytoskeletal dynamics, transcription, protein synthesis, metabolic responses and membrane trafficking [1–4]. PI3Ks are recruited and activated down- stream of tyrosine kinase receptors (RTKs) and G-protein- coupled receptors (GPCRs) and phosphorylate phospho- inositides (PIs) in their D3 position. Three different PI3K classes can be distinguished based on their substrate specificity and molecular structure (Table 1). Class I PI3Ks transduce responses to agonists by regulating the cellular levels of phosphoinositide (3,4,5)-trisphosphate (PIP 3 ), whereas class II and III PI3Ks use PI as a substrate to produce phosphoinositide 3-phosphate (PI3P) [5]. In class I PI3Ks, the lipid kinase activity is crucially controlled by protein–protein interactions. These PI3Ks function as obligate heterodimers composed of a catalytic and an adaptor/regulatory subunit * . Mammals express four catalytic subunits (p110a,-b,-g and -d) that share structural homology and that each contain an adaptor- binding domain, a Ras-binding domain (RBD), a C2 domain potentially involved in membrane attachment, a helical/accessory domain (PIK) and a kinase domain (cat- alytic domain). Each catalytic p110 subunit binds directly to a regulatory subunit (where p85a, p85b, p55a, p55g or p50a can complex with either p110a,-b or -d; and where p101 or p84/87 y can complex with p110g)(Table 1) [5]. Association of the catalytic subunit to an adaptor/regulator modifies its subcellular localization and enzymatic activity. For instance, binding of p110 to p85 inhibits its catalytic activity in the cytoplasm. Conversely, through its SH2 domain, the p85 isoforms tether the complex to acti- vated RTKs. This interaction not only releases the inhi- bition on p110 but also localizes this subunit next to its lipid substrates at the membrane [5]. It has been recently reported that a novel protein, PI3K-interacting protein 1 (PIK3IP1), interacts with p110a and -b, acting as another regulator of p110 function [6]. Furthermore, p110 activity is modulated by its association with Ras, which interacts with the RBD domain. The selective binding of GTP–Ras to p110a,-g or -d is thought to trigger a positive-feedback loop, generating a synergy between Ras and PI3K sig- naling [7]. Most of the structural details explaining the regulation of p110 catalytic function exerted by its association with p85 isoforms or Ras have been elucidated [8,9]. However, the abundance of solvent-exposed surfaces on p110a and p110g crystals suggests further protein–protein inter- actions [10,11]. For instance, recent evidence has been provided that p110g interacts with protein kinase Ca (PKCa) [12]. In this context, p110g phosphorylates PKCa and modulates its activity. This can be considered as a kinase–substrate interaction, but it has recently emerged that p110 subunits can also act, independently of their kinase activity, as scaffold proteins. This indicates a novel layer of complexity in p110-mediated signaling, which depends on protein–protein interactions and does not involve the modulation of the catalytic function of PI3Ks. Indeed, a specific kinase-independent role was first reported for p110g, and a distinct selective scaffolding function has recently been described for p110b as well. Kinase-independent roles of p110g p110g is activated by G bg subunits of GPCRs. The bio- logical roles of this class I PI3K have been well character- ized in hematopoietic cells, where p110g activates a downstream signaling pathway essential for the regulation of immunity and inflammation [2]. Accordingly, p110g-null mice present reduced leukocyte migration towards inflam- matory sites, which is phenocopied by mice that express a kinase-dead p110g (p110g KD/KD ) [13]. Furthermore, selec- tive p110g inhibitors are anti-inflammatory in multiple disease models [14,15]. A twist in our understanding of p110g signaling has come from studies on cardiomyocyte biology. Remarkably, p110g is expressed also in the heart, where it regulates b-adrenergic signaling in a kinase-dependent manner [16]. Both lipid and protein Review Corresponding author: Hirsch, E. (emilio.hirsch@unito.it) * p85 functions as both an adaptor and a regulator.. y The double name indicates different molecular weights found for the same protein.. 244 0968-0004/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2009.02.003 Available online 17 April 2009