Class I phosphoinositide 3-OH kinase (PI3K) signaling is central to animal growth and metabolism, and pathological disruption of this pathway affects cancer and diabetes. However, the specific spatial/temporal dynamics and signaling roles of its minor lipid messenger, phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2), are not well understood. This owes principally to a lack of tools to study this scarce lipid. Here we developed a high-sensitivity genetically encoded biosensor for PI(3,4)P2, demonstrating high selectivity and specificity of the sensor for the lipid. We show that despite clear evidence for class II PI3K in PI(3,4)P2-driven function, the overwhelming majority of the lipid accumulates through degradation of class I PI3K-produced PIP3. However, we show that PI(3,4)P2 is also subject to hydrolysis by the tumor suppressor lipid phosphatase PTEN. Collectively, our results show that PI(3,4)P2 is potentially an important driver of class I PI3K-driven signaling and provides powerful new tools to begin to resolve the biological functions of this lipid downstream of class I and II PI3K.
STIM1 and Orai1 are the main components of a widely conserved Calcium influx pathway known as store-operated calcium entry (SOCE). STIM1 is a calcium sensor, which oligomerizes and activates Orai channels when calcium levels drop inside the endoplasmic reticulum (ER). The series of molecular rearrangements that STIM1 undergoes until final activation of Orai1 require the direct exposure of the STIM1 domain known as SOAR (Stim Orai Activating Region). In addition to these complex molecular rearrangements, other constituents like lipids at the plasma membrane, play critical roles orchestrating SOCE. PI(4,5)P2 and enriched cholesterol microdomains have been shown as important signaling platforms that recruit the SOCE machinery in steps previous to Orai1 activation. However, little is known about the molecular role of cholesterol once SOCE is activated. In this study we provide clear evidence that STIM1 has a cholesterol-binding domain located inside the SOAR region and modulates Orai1 channels. We demonstrate a functional association of STIM1 and SOAR to cholesterol, indicating a close proximity of SOAR to the inner layer of the plasma membrane. In contrast, the depletion of cholesterol induces the SOAR detachment from the plasma membrane and enhances its association to Orai1. These results are recapitulated with full length STIM1.
Store Operated Calcium Entry (SOCE) is one of the most important mechanisms for calcium mobilization in to the cell. Two main proteins sustain SOCE: STIM1 that acts as the calcium sensor in the endoplasmic reticulum (ER) and Orai1 responsible for calcium influx upon depletion of ER. There are many studies indicating that SOCE is modulated by the cholesterol content of the plasma membrane (PM). However, a myriad of questions remain unanswered concerning the precise molecular mechanism by which cholesterol modulates SOCE.
In the present study we found that reducing PM cholesterol results in the internalization of Orai1 channels, which can be prevented by overexpressing caveolin 1 (Cav1). Furthermore, Cav1 and Orai1 associate upon SOCE activation as revealed by FRET and coimmunoprecipitation assays. The effects of reducing cholesterol were not limited to an increased rate of Orai1 internalization, but also, affects the lateral movement of Orai1, inducing movement in a linear pattern (unobstructed diffusion) opposite to basal cholesterol conditions were most of Orai1 channels moves in a confined space, as assessed by Fluorescence Correlation Spectroscopy, Cav1 overexpression inhibited these alterations maintaining Orai1 into a confined and partially confined movement.
These results not only highlight the complex effect of cholesterol regulation on SOCE, but also indicate a direct regulatory effect on Orai1 localization and compartmentalization by this lipid.
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