Using structure to define the function of phosphoinositide 3‐kinase family members

Abstract

It is now clear that lipid phosphorylation and hydrolysis plays a pivotal role in the transmission of extracellular stimuli across the cytoplasm [1, 2]. Consequently, great effort has been applied to characterize the enzymes responsible for these signalling events [3-5]. This article will present an overview of one family of lipid kinases; namely the phosphoinositide 3-kinases (PI3-kinases) which are found in species ranging from slime mold to man and catalyse the addition of phosphate to the 3′ position on the inositol ring of phosphoinositides. We offer a classification of the rapidly expanding number of PI3-kinase family members based principally on their structure and discuss our understanding of their regulation and downstream targets. In this way we hope to provide a framework within which the nomenclature of PI3-kinase family members can be standardised. In the mid 1980's immunoprecipitates of pp60v-src, polyoma middle T/pp60c-src complexes and p68v-ros, the transforming protein of the avian sarcoma virus UR2 were found to contain a novel lipid kinase activity which phosphorylated phosphatidylinositol (PtdIns) on the 3′ position of its inositol ring (reviewed in [6]). A similar PI3-kinase activity was later found associated with activated platelet derived growth factor receptors where PtdIns(4)P and PtdIns(4,5)P2 were used as substrate [7]. None of these 3′ phosphorylated lipids served as substrates for phospholipase C or D suggesting that they could fulfil an independent second messenger function [8]. Such findings stimulated great excitement since they suggested that pharmaceutical attenuation of this novel lipid kinase activity could inhibit mitogenesis and consequently control oncogenesis. Numerous studies have subsequently demonstrated a much broader role for this lipid kinase and its products in cell physiology/ pathophysiology [4]. At the time it was unclear as to how one enzyme could mediate such a multiplicity of functions. A clue as to how such specificity could be achieved was however, already evident from those early experiments. Ligand stimulation acutely elevated levels of both PtdIns(3,4)P2 and PtdIns(3,4,5)P3 compared to resting cells (reviewed in [9]). In contrast, the constitutively elevated level of PtdIns(3)P remained largely unaltered. Such results suggested that PtdIns(3)P plays a more central, metabolic role whilst the more highly phosphorylated phosphoinositides function as the ligand responsive second messengers. PI3-kinase was first identified as an 85-kDa phosphoprotein whose appearance correlated with a PI3-kinase activity in immunoprecipitates from a number of polyoma middleT mutants [10]. These studies facilitated the purification and subsequent cloning of a heterodimeric complex comprised of an 85-kDa adaptor (p85) and a 110-kDa catalytic subunit (p110) [11, 12]. Cloning of the PI3-kinase catalytic subunit revealed a sequence similarity within its catalytic domain to yeast Vps34p a protein involved in vesicular trafficking later shown to also possess PI3-kinase activity [13]. Such sequence alignment within the kinase domains allowed the use of polymerase chain reaction based strategies to isolate and characterize novel PI3-kinase family members. Over the last five years we have witnessed an explosion in the number of PI3-kinase catalytic and regulatory subunits described in the literature [5]. Consequently, the wealth of information generated has proved confusing for many unfamiliar with this area of study. Fortunately, as we learn more about this family of signalling molecules, a basis for their classification has emerged. The classification of PI3-kinase family members we propose is based on their structure and probable mechanism of regulation. In mammals, we currently know of 7 forms of the catalytic subunit and 5 forms of associated binding partner /adaptor subunit. These catalytic and adaptor subunits can be divided into three main classes (Fig. 1 ): These enzymes all form a heterodimeric complex with an adaptor protein which renders them responsive to ligand stimulation. In vitro, they can utilise PtdIns, PtdIns(4)P and PtdIns(4,5)P2 as substrate but in the cell, they appear to preferentially phosphorylate PtdIns(4,5)P2 [9]. The reason for this disparity is presently unclear although it is most likely to be a consequence of substrate presentation (see below). This class of PI3-kinase is subdivided based on the form of adaptor subunit with which the catalytic subunit associates. These form a heterodimeric complex consisting of a 110–120-kDa catalytic subunit and an adaptor protein which contains two src homology 2 (SH2) domains. Mammalian class IA catalytic subunits include p110α, p110β and p110δ [14-16]. They all contain a domain which binds p85, a region for ras association [17] a PI kinase (PIK) domain and a C-terminal catalytic domain. The PIK domain is a region conserved amongst all PI3 and PI4-kinases and although its role is presently unclear, it is likely t