Phosphoinositide Kinases Play Key Roles in Norepinephrine- and Angiotensin II-induced Increase in Phosphatidylinositol 4,5-Bisphosphate and Modulation of Cardiac Function

Gulyás

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

et al. 2016

Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay. We demonstrate robust and sensitive measurements of PtdIns4P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics, as well as changes in cytoplasmic Ins(1,4,5)P3 levels. Measurements were made during either experimental activation of lipid degradation, or PI 3-kinase and phospholipase C mediated signal transduction. Our results reveal a previously unappreciated synthesis of PtdIns4P that accompanies moderate activation of phospholipase C signaling downstream of both EGF and muscarinic M3 receptor activation. This signaling-induced PtdIns4P synthesis relies on protein kinase C, and implicates a feedback mechanism in the control of inositol lipid metabolism during signal transduction.

Section: Discussionmentioning

confidence: 99%

BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation

Gulyás

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

et al. 2016

“…1 C). At this high concentration, wortmannin is known to block PI4K [26] and thus PI4K rather than PI3K activity might be implied. Therefore, we used a PI4K inhibitor, PAO to see if PI4K was involved in IP 7 regulation.…”

Section: Resultsmentioning

confidence: 98%

Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic β-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity

Rajasekaran

Illies

Shears

et al. 2018

Cellular Signalling

Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP7, is at a high level in pancreatic β-cells and is important for insulin exocytosis. To better understand IP7 regulation in β-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP7. Although the inhibitors have diverse targets, they all perturbed IP7 levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP7 levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high Km for ATP makes IP7 synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP7 concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wortmannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (U73122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP7 indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic β-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP7’s role in cellular signal transduction is likely to have been underestimated.

American Journal of Physiology-Heart and Circulatory Physiology

“…Some signaling pathways, such as PKC and PI4K, were involved in producing PIP 2 in myocytes isolated from adult mice treated with ANG II (40). In addition, the PIP 2 level could be converted at plasma membrane to PIP 3 by PTEN, which is one of the regulators of PIP 2 located there.…”

Section: Discussionmentioning

confidence: 96%

“…PIP 2 was localized to the Z-disc, which suggested its role in sarcomeric reorganization (14). Furthermore, PIP 2 is implicated in ANG II-induced cardiac hypertrophy in an animal model (40).…”

mentioning

confidence: 99%

Actin dynamics is rapidly regulated by the PTEN and PIP₂ signaling pathways leading to myocyte hypertrophy

Tanhehco

Russell

2014