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.
Receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) stimulate phosphoinositide 3-kinases (PI3Ks) to convert phosphatidylinositol-4,5-bisphosophate [PtdIns(4,5)P] into phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P]. PtdIns(3,4,5)P then remodels actin and gene expression, and boosts cell survival and proliferation. PtdIns(3,4,5)P partly achieves these functions by triggering activation of the kinase Akt, which phosphorylates targets like Tsc2 and GSK3β. Consequently, unchecked upregulation of PtdIns(3,4,5)P-Akt signalling promotes tumour progression. Interestingly, 50-70% of PtdIns and PtdInsPs have stearate and arachidonate at -1 and -2 positions of glycerol, respectively, forming a species known as 38:4-PtdIns/PtdInsPs. LCLAT1 and MBOAT7 acyltransferases partly enrich PtdIns in this acyl format. We previously showed that disruption of LCLAT1 lowered PtdIns(4,5)P levels and perturbed endocytosis and endocytic trafficking. However, the role of LCLAT1 in receptor tyrosine kinase and PtdIns(3,4,5)P signaling was not explored. Here, we show that LCLAT1 silencing in MDA-MB-231 and ARPE-19 cells abated the levels of PtdIns(3,4,5)P in response to EGF signalling. Importantly, LCLAT1-silenced cells were also impaired for EGF-driven and insulin-driven Akt activation and downstream signalling. Thus, our work provides first evidence that the LCLAT1 acyltransferase is required for receptor tyrosine kinase signalling.
The PIP/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP/PI(3,4)P phosphatase, PTEN. Despite huge research investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signaling and constrained by pathway feedback. In the absence of PTEN, the network is dramatically remodeled. A poorly understood YXXM- and PIP/PI(3,4)P-binding PH domain-containing adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP, AKT phosphorylation, and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K- and Src-family kinase-dependent phosphorylation of YXXM, eliciting PI3K activation. hPLEKHS1 mRNA and activating Y phosphorylation of hSrc correlated with PI3K pathway activity in human prostate cancers. We propose that in PTEN-null cells receptor-independent, Src-dependent tyrosine phosphorylation of PLEKHS1 creates positive feedback that escapes homeostasis, drives PIP signaling, and supports tumor progression.
Myeloproliferative neoplasms (MPNs) are characterized by the activated JAK2-STAT pathway. Pleckstrin-2 (Plek2) is a downstream target of the JAK2-STAT pathway and overexpressed in patients with MPNs. We previously revealed that Plek2 plays critical roles in the pathogenesis of JAK2 mutated MPNs. The non-essential roles of Plek2 under physiologic conditions makes it an ideal target for MPN therapy. Here we identified first-in-class Plek2 inhibitors through an in silico high-throughput screening and cell-based assays followed by the synthesis of analogs. The Plek2 specific small molecule inhibitors showed potent inhibitory effects on cell proliferation. Mechanistically, Plek2 interacts with and enhances the activity of Akt through the recruitment of downstream effector proteins. The Plek2 signaling complex also includes Hsp72 that protects Akt from degradation. These functions were blocked by Plek2 inhibitors via their direct binding to Plek2 DEP domain. The role of Plek2 in activating the Akt signaling was further confirmed in vivo using a hematopoietic specific Pten knockout mouse model. We next tested Plek2 inhibitors alone or in combination with an Akt inhibitor in various MPN mouse models, which showed significant therapeutic efficacies similar to the genetic depletion of Plek2. The Plek2 inhibitor was also effective in reducing proliferation of CD34 positive cells from MPN patients. Our studies reveal a Plek2-Akt complex that drives cell proliferation and can be targeted by a new class of anti-proliferative compounds for MPN therapy.