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Content archived on 2024-05-29

The roles of 3-kinase isoforms in cell migration

Final Activity Report Summary - PI3K IN MIGRATION (The roles of 3-kinase isoforms in cell migration)

Downstream of tyrosine kinases and Ras, PIP3 is produced by the class IA subset of PI3Ks. These are heterodimers made up of a 110 kDa catalytic subunit (p110a, p110b and p110d) in complex with one of 5 regulatory subunits (collectively called the 'p85s'). Whereas p110aand p110b are found in most tissues, the expression of p110d is most abundant in leukocytes.

In the present project, we have used genetic and pharmacological approaches to dissect the mechanism of p110d and p110a action. Primary macrophages were used as a main cell model, and were derived either from wild-type (WT) mice or from mice in which both p110d alleles are replaced by a kinase-dead version of p110d (called p110dD910A), as a result of knock-in mouse gene targeting1. Bone marrow-derived macrophages (BMMs) from these mice are further referred to as dD910A/D910A cells. In order to assess the dose-dependency of p110 isoform function2, we also derived BMMs from heterozygous p110dD910A or p110aD933A mice (the latter express a kinase-dead version of p110a p110aD933A/D933A mice are embryonic lethal3). These heterozygous BMMs are further referred to as dD910A/WT and aD933A/WT, respectively. Pharmacological intervention with p110d was achieved by the use of IC87114, a small molecule inhibitor with selectivity for p110d.

PIP3 action in cells is counteracted by lipid phosphatases, amongst which PTEN is the most extensively investigated. Indeed, PTEN is mutationally inactivated in a wide variety of cancers, contributing to the constitutive activation of the PI3K pathway in these cells5-8. This is thought to be a unidirectional relationship in which PTEN degrades the lipids produced by PI3K, thus controlling cell proliferation, survival and migration. We now show that this relationship is in fact bidirectional, whereby PI3K reciprocally controls PTEN. During the Marie Curie fellowship we have discovered that the p110d?PI3K negatively regulates PTEN, through a pathway involving inhibition of RhoA. Inactivation of p110d in macrophages led to reduced Akt and Rac1 activation after CSF-1 stimulation, but paradoxically, to increased RhoA and PTEN activity even under basal conditions. Pharmacological inhibition of ROCK, a downstream effector kinase of RhoA, restored all signalling and functional defects of p110d inactivation, including Akt phosphorylation, chemotaxis and proliferation. This work identifies the RhoA/ROCK pathway as a major target of p110d-mediated PI3K signalling, and establishes for the first time that PI3K controls itself, via a feedback loop involving PTEN.