Life Sciences Research for Lifelong Health

Len Stephens

Research Summary

The programmes of work in the laboratory are currently aimed at understanding the molecular mechanisms and physiological significance of intracellular signalling networks which involve a family of enzymes called phosphoinositide 3OH-kinases (PI3Ks).

PI3Ks are now accepted to be critical regulators of numerous important and complex cell responses, including cell growth, division, survival and movement.

PI3Ks catalyse the formation of one or more critical phospholipid messenger molecules, which signal information by binding to specific domains in target proteins. Currently the best understood pathway involves the activation of Class I PI3Ks by cell surface receptors.

In recent years, the laboratory has increasingly focused on the role of PI3Ks in the signalling mechanisms which allow receptors on neutrophils (white blood cells) to control various aspects of neutrophil function.

Neutrophils are key players in the front line of our immune system, responsible primarily for the recognition and destruction of bacterial and fungal pathogens. However, they are also involved in the amplification cascades that underlie various inflammatory pathologies, e.g. Acute Respiratory Distress Syndrome (ARDS) and rheumatoid arthritis.

Latest Publications

In B cells, phosphatidylinositol 5-phosphate 4-kinase-α synthesizes PI(4,5)P2 to impact mTORC2 and Akt signaling.
Bulley SJ, Droubi A, Clarke JH, Anderson KE, Stephens LR, Hawkins PT, Irvine RF

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are enigmatic lipid kinases with physiological functions that are incompletely understood, not the least because genetic deletion and cell transfection have led to contradictory data. Here, we used the genetic tractability of DT40 cells to create cell lines in which endogenous PI5P4Kα was removed, either stably by genetic deletion or transiently (within 1 h) by tagging the endogenous protein genomically with the auxin degron. In both cases, removal impacted Akt phosphorylation, and by leaving one PI5P4Kα allele present but mutating it to be kinase-dead or have PI4P 5-kinase activity, we show that all of the effects on Akt phosphorylation were dependent on the ability of PI5P4Kα to synthesize phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] rather than to remove PI5P. Although stable removal of PI5P4Kα resulted in a pronounced decrease in Akt phosphorylation at Thr308 and Ser473, in part because of reduced plasma membrane PIP3, its acute removal led to an increase in Akt phosphorylation only at Ser473. This process invokes activation primarily of mammalian target of rapamycin complex 2 (mTORC2), which was confirmed by increased phosphorylation of other mTORC2 substrates. These findings establish PI5P4Kα as a kinase that synthesizes a physiologically relevant pool of PI(4,5)P2 and as a regulator of mTORC2, and show a phenomenon similar to the "butterfly effect" described for phosphatidylinositol 3-kinase Iα [Hart JR, et al. (2015) Proc Natl Acad Sci USA 112(4):1131-1136], whereby through apparently the same underlying mechanism, the removal of a protein's activity from a cell can have widely divergent effects depending on the time course of that removal.

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Proceedings of the National Academy of Sciences of the United States of America, , 1091-6490, , 2016

PMID: 27601656

Phosphoproteomic Analyses of Interleukin 2 Signaling Reveal Integrated JAK Kinase-Dependent and -Independent Networks in CD8(+) T Cells.
Ross SH, Rollings C, Anderson KE, Hawkins PT, Stephens LR, Cantrell DA

Interleukin-2 (IL-2) is a fundamental cytokine that controls proliferation and differentiation of T cells. Here, we used high-resolution mass spectrometry to generate a comprehensive and detailed map of IL-2 protein phosphorylations in cytotoxic T cells (CTL). The data revealed that Janus kinases (JAKs) couple IL-2 receptors to the coordinated phosphorylation of transcription factors, regulators of chromatin, mRNA translation, GTPases, vesicle trafficking, and the actin and microtubule cytoskeleton. We identified an IL-2-JAK-independent SRC family Tyr-kinase-controlled signaling network that regulates ∼10% of the CTL phosphoproteome, the production of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), and the activity of the serine/threonine kinase AKT. These data reveal a signaling framework wherein IL-2-JAK-controlled pathways coordinate with IL-2-independent networks of kinase activity and provide a resource toward the further understanding of the networks of protein phosphorylation that program CTL fate.

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Immunity, , 1097-4180, , 2016

PMID: 27566939

Coincident signals from GPCRs and receptor tyrosine kinases are uniquely transduced by PI3Kβ in myeloid cells.
Houslay DM, Anderson KE, Chessa T, Kulkarni S, Fritsch R, Downward J, Backer JM, Stephens LR, Hawkins PT

Class I phosphoinositide 3-kinases (PI3Ks) catalyze production of the lipid messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3), which plays a central role in a complex signaling network regulating cell growth, survival, and movement. This network is overactivated in cancer and inflammation, and there is interest in determining the PI3K catalytic subunit (p110α, p110β, p110γ, or p110δ) that should be targeted in different therapeutic contexts. Previous studies have defined unique regulatory inputs for p110β, including direct interaction with Gβγ subunits, Rac, and Rab5. We generated mice with knock-in mutations of p110β that selectively blocked the interaction with Gβγ and investigated its contribution to the PI3K isoform dependency of receptor tyrosine kinase (RTK) and G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) responses in primary macrophages and neutrophils. We discovered a unique role for p110β in supporting synergistic PIP3 formation in response to the coactivation of macrophages by macrophage colony-stimulating factor (M-CSF) and the complement protein C5a. In contrast, we found partially redundant roles for p110α, p110β, and p110δ downstream of M-CSF alone and a nonredundant role for p110γ downstream of C5a alone. This role for p110β completely depended on direct interaction with Gβγ, suggesting that p110β transduces GPCR signals in the context of coincident activation by an RTK. The p110β-Gβγ interaction was also required for neutrophils to generate reactive oxygen species in response to the Fcγ receptor-dependent recognition of immune complexes and for their β2 integrin-mediated adhesion to fibrinogen or poly-RGD+, directly implicating heterotrimeric G proteins in these two responses.

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Science signaling, 9, 1937-9145, ra82, 2016

PMID: 27531651

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Latest Publications

In B cells, phosphatidylinositol 5-phosphate 4-kinase-α synthesizes PI(4,5)P2 to impact mTORC2 and Akt signaling.

Bulley SJ, Droubi A, Clarke JH

Proceedings of the National Academy of Sciences of the United States of America
1091-6490: (2016)

PMID: 27601656

Coincident signals from GPCRs and receptor tyrosine kinases are uniquely transduced by PI3Kβ in myeloid cells.

Houslay DM, Anderson KE, Chessa T

Science signaling
9 1937-9145:ra82 (2016)

PMID: 27531651

The inositol-3-phosphate synthase biosynthetic enzyme has distinct catalytic and metabolic roles.

Frej AD, Clark J, Roy CL

Molecular and cellular biology
1098-5549: (2016)

PMID: 26951199

Emerging evidence of signalling roles for PI(3,4)P2 in Class I and II PI3K-regulated pathways.

Hawkins PT, Stephens LR

Biochemical Society transactions
44 1470-8752:307-14 (2016)

PMID: 26862220

Inactivation of the Class II PI3K-C2β Potentiates Insulin Signaling and Sensitivity.

Alliouachene S, Bilanges B, Chicanne G

Cell reports
13 2211-1247:1881-94 (2015)

PMID: 26655903

Investigating the effect of arachidonate supplementation on the phosphoinositide content of MCF10a breast epithelial cells.

Anderson KE, Juvin V, Clark J

Advances in biological regulation
2212-4934: (2015)

PMID: 26639089

Localizing the lipid products of PI3Kγ in neutrophils.

Norton L, Lindsay Y, Deladeriere A

Advances in biological regulation
2212-4934: (2015)

PMID: 26596865

The cytotoxic T cell proteome and its shaping by the kinase mTOR.

Hukelmann JL, Anderson KE, Sinclair LV

Nature immunology
1529-2916: (2015)

PMID: 26551880

The regulatory subunits of PI3Kγ control distinct neutrophil responses.

Deladeriere A, Gambardella L, Pan D

Science signaling
8 1937-9145:ra8 (2015)

PMID: 25605974

PI3K signalling in inflammation.

Hawkins PT, Stephens LR

Biochimica et biophysica acta
1851 0006-3002:882-97 (2015)

PMID: 25514767