Development and characterisation of tetracycline-regulated phosphoinositide 3-kinase mutants: assessing the role of multiple phosphoinositide 3-kinases in chemokine signaling.

A combination of pharmacological, biochemical, molecular and genetic evidence supports a key role for phosphoinositide 3-kinase (PI3K) and its associated signalling cascade in cell migration in response to members of the chemokine family. PI3Ks can be divided into three main classes on the basis of their in vitro lipid substrate specificity, structure and likely mode of regulation. The prototypical class I PI3Ks are heterodimers consisting of the class I(A) 85-kDa regulatory/adaptor subunit and a catalytic 110-kDa subunit and the class I(B) PI3K (PI3Kgamma), which is stimulated by G protein betagamma subunits. Whilst genetic evidence supports a key role for PI3Kgamma in mediating chemotactic responses, it is clear that other PI3K isoforms can be activated by chemokines and can potentially contribute to the chemotactic responses to chemokines. In order to get a more accurate picture of the precise role of individual PI3Ks in functional responses to chemokines, we report development of tetracycline-inducible dominant-negative constructs of the class I(A) and class I(B) PI3Ks and their expression in the leukemic T cell line Jurkat. SDF-1/CXCR4-mediated chemotaxis of Jurkat cells is strongly, but incompletely abrogated (e.g. approximately 60-70%) in clones expressing the dominant-negative PI3Kgamma construct. Interestingly, Jurkat cells expressing a dominant-negative mutant of class I(A) PI3K also exhibited marked abrogation of chemotactic responses to SDF-1, albeit to lesser extent (e.g. approximately 30-40% inhibition) than observed with the class I(B) mutant. These data suggests that both class I(A) and class I(B) isoforms can contribute to chemotactic responses, and both are required for optimal migratory responses to SDF-1. Furthermore, neither isoform alone is able to support optimal migration in the absence of the other. This may reflect an important interplay between the two different forms of PI3K that has yet to be fully elucidated. The use of inducible expression systems such as that described here will be an important approach in assessing the role of not only individual PI3Ks, but also their downstream effector proteins, in supporting actin polymerisation and cytoskeletal rearrangements as well as chemotaxis and adhesion molecule up-regulation. Copyright 2002 Elsevier Science B.V.