Phosphatidylinositol 3kinases

The PI3Ks are enzymes that are grouped into three classes (I-III). Most members of this family are bound to regulatory subunits which determine its specificty and function [83-85]. Class I PI3Ks are subdivided into IA and IB and are members to the canonical PI3K/AKT pathway. They are heterodimeric serine and threonine kinases comprising a catalytic subunit, p110, and a regulatory subunit encoded by the PIK3CA and PIK3R1 genes, respectively [83]. The four isoforms of p110 (a-5) and their regulatory subunits have distinct structure-function domains and specificity. For p110a, and 5 the most commonly associated regulatory subunit has been identified as p85 [83]. Specific isoforms of the p85 adaptor subunit (p85a, p85|, p50a, p55a, or p55y) facilitate the interaction with RTKs as well as the p110 catalytic domain isoforms [86]. The p85 subunit directly associates with active RTKs through the physical interaction of its SRC homology 2 (SH2) domain at phosphotyrosine residues of RTKs [87]. The consensus sequence has been identified to be YXXM [87]. In particular, Class IA PI3Ks' p85a subunit encodes an adaptor-like protein that has two SH2 domains and an inter-SH2 domain that binds constitutively to the p110 catalytic subunit [87]. The two splice variants (p55a and p50a) retain such regions but lack an amino terminal SH3 domain (mediates the binding of proline rich sequences) and a breakpoint cluster region (BCR) homology domain (a protein-protein interaction motif) [87]. The p110 isoforms (a, 5) have the same basic structure, including a kinase domain and a C2 domain for membrane anchoring [87].

Class I PI3Ks, once activated by RTKs (Class IA) or G-protein-coupled receptors (GPCRs, [Class IB]), have preferred substrates, in particular, the non-phosphorylated phosphatidylinositol (PI), inositol monophosphate (PI(4)P), and phosphatidyl-bis-phosphate (PI(4,5)P2), and mediate the addition of a phosphate group in the D-3 position of the inositol ring to generate PI(3)P, PIP2 and PIP3, respectively [83, 88]. PIP3 is a potent second messenger in the cell and the predominant arbitrator of PI3K signalling. Class IA PI3K p110a domain isoform is the most mutated amongst cancers, causing the kinase to be more active [86, 89, 90] and perpetuating a constitutively active PI3K pathway. Class II and III PI3Ks, on the other hand, are less studied and are recognized for their involvement in membrane trafficking and receptor internalization, and, vesicle trafficking, respectively [91-93]. PI3Ks within Class II generate PI(3,4)P2 from PI(3)P and can also produce PI(3)P from PI. However, they cannot recognize PIP(4,5)P2 as substrate to produce PIP and PIP2. Class II PI3Ks use only PI to convert it to PIP [83, 94]. Furthermore, unlike Class I PI3Ks, Class II PI3Ks do not require a regulatory subunit but comprise three distinct isoforms to mediate their functions. Class IA PI3K will be discussed in this review and will be referred to as PI3K unless otherwise stated.

PI3K activity is normally strictly regulated within the cell by growth factor-receptor interactions [95]. As such, the majority of the PI3K is inactive in the cytosplasm and remains removed from its plasma membrane substrates. Moreover, only a small fraction of these PI3Ks become activated upon growth factor stimulation [95]. Currently, it is thought that there are preformed inactive p85-p110 complex present in the cytoplasm, whereby ligand mediated activation of kinase activity and transphosphorylation of RTK's cytoplasmic tail recruits p85-p110 complexes to the receptor by the SH2 domain of p85 [87]. This brings PI3K in close proximity to its lipid substrates; moreover, it is reasoned that the RTK-p85 interaction may remove an inhibitory effect of p85 on p110 kinase activity [87, 96]. This is thought to involve conformational changes in the p85-p110 complex through the SH3 and BCR domains.

Mutations have now been identified in the genes coding for the p110 and p85 subunits which have shed light on the pathology of metabolic diseases and cancer [83, 93]. These mutations occur at a frequency of 5-25% in common cancers such as breast, endometrium and large intestine [83]. Activating mutations or 'hot spots' of PIK3CA occur at a frequency of 80% and are located in the PI3K catalytic kinase domain, H1074 and the helical domain, E542 and E545 [93]. Both mutations have been demonstrated to drive transformation in vitro [93, 97]. As a result, the lipid kinase activity is increased [83, 89, 98-102], downstream signalling no long requires upstream growth factor stimulation, and increased oncogenic potency. Expression of these hot spot mutants induced oncogenic transformation in avian and mammalian cell culture and transgenic expression of H1047R p110a in mouse models induced adenocarcinoma of the lung [83, 103]. As such, hot spot mutations then can be suggested to function as drivers of cellular transformation to a more oncogenic phenotype. Conversely, mutational inactivation of the ability of p110a to interact with RAS has the opposite effect by decreasing the oncogenicity of helical domain mutants and minimizing downstream signaling [83]. On the other hand, kinase domain mutants become independent of RAS binding, and its oncogenicity is preserved [83]. PIK3R1 mutations occur within a stretch of six residues (560-565) located in the inter-SH2 domain of p85 [83]. This area is the contact point for p85 with the C2 domain of p110a whereby mutation leads to improper binding to p110a and relieve the inhibitory interaction of p85 [83]. Enhanced AKT signalling, stimulation of cell replication, and oncogenic transformation were some of the observed effects [83, 104, 105]. As such, p85 mutations in the inter-SH2 domain can be thought to be equivalent to activation mutations of the p110a C2 domain.

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