Lisa M. Ballou

Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function

A critical regulator of autophagy is the Class III PI3K Vps34 (also called PIK3C3). Although Vps34 is known to play an essential role in autophagy in yeast, its role in mammals remains elusive. To elucidate the physiological function of Vps34 and to determine its precise role in autophagy, we have generated Vps34f/f mice, in which expression of Cre recombinase results in a deletion of exon 4 of Vps34 and a frame shift causing a deletion of 755 of the 887 amino acids of Vps34. Acute ablation of Vps34 in MEFs upon adenoviral Cre infection results in a diminishment of localized generation of phosphatidylinositol 3-phosphate and blockade of both endocytic and autophagic degradation. Starvation-induced autophagosome formation is blocked in both Vps34-null MEFs and liver. Liver-specific Albumin-Cre;Vps34f/f mice developed hepatomegaly and hepatic steatosis, and impaired protein turnover. Ablation of Vps34 in the heart of muscle creatine kinase-Cre;Vps34f/f mice led to cardiomegaly and decreased contractility. In addition, while amino acid-stimulated mTOR activation was suppressed in the absence of Vps34, the steady-state level of mTOR signaling was not affected in Vps34-null MEFs, liver, or cardiomyocytes. Taken together, our results indicate that Vps34 plays an essential role in regulating functional autophagy and is indispensable for normal liver and heart function.

Rapamycin and mTOR kinase inhibitors

Mammalian target of rapamycin (mTOR) is a protein kinase that controls cell growth, proliferation, and survival. mTOR signaling is often upregulated in cancer and there is great interest in developing drugs that target this enzyme. Rapamycin and its analogs bind to a domain separate from the catalytic site to block a subset of mTOR functions. These drugs are extremely selective for mTOR and are already in clinical use for treating cancers, but they could potentially activate an mTOR-dependent survival pathway that could lead to treatment failure. By contrast, small molecules that compete with ATP in the catalytic site would inhibit all of the kinase-dependent functions of mTOR without activating the survival pathway. Several non-selective mTOR kinase inhibitors have been described and here we review their chemical and cellular properties. Further development of selective mTOR kinase inhibitors holds the promise of yielding potent anticancer drugs with a novel mechanism of action.

PI 3-kinase regulation of dopamine uptake

The magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3‐kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [3H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3‐kinase, increased [3H]DA uptake and blocked the amphetamine‐induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3‐kinase. The LY294002‐induced reduction in [3H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin‐dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs.

Suppression of Phosphoinositide 3-Kinase Signaling and Alteration of Multiple Ion Currents in Drug-Induced Long QT Syndrome

The dangerous heart arrhythmias that are triggered as a side effect of some drugs are caused by many ion channels, prompting a rethinking of how we screen for these adverse events. Long QT: A Many-Channeled Syndrome To “do no harm” is a key principle of medical ethics, yet the use of some drugs can trigger life-threatening side effects. For example, the anticancer drug nilotinib—a tyrosine kinase inhibitor—can cause sudden death by inducing an irregular heartbeat and, as such, carries a black box warning from the U.S. Food and Drug Administration. Specifically, nilotinib (and other medications) can produce long QT syndrome, in which repolarization of the heart is delayed after a heartbeat. This effect is believed to be caused by direct blockade of the potassium ion channel through which the repolarizing current flows. Because some phosphoinositide 3-kinases (PI3Ks)—intracellular signal-transducing enzymes—are activated by tyrosine kinases, Lu et al. investigated whether the cardiac effects of nilotinib and related drugs might in part be mediated by PI3Ks. In isolated cardiac cells, delayed repolarization is seen as an increase in action potential duration (APD); as expected, treatment with these drugs increased the APD, whereas PI3K activity decreased. (Likewise, a PI3K inhibitor increased the APD.) The addition of the second messenger produced by PI3K normalized the APD, indicating that drug-induced PI3K inhibition is responsible for the increased APD. Lu et al. also showed that both nilotinib and a PI3K inhibitor affected currents through multiple ion channels, including calcium and sodium channels, in addition to the potassium channel originally thought to be responsible for drug-induced long QT syndrome. Furthermore, isolated mouse hearts lacking a PI3K subunit displayed a prolonged QT interval on an electrocardiogram—the sign of long QT syndrome. Although nilotinib increased this interval in wild-type hearts, it had no effect on those lacking the PI3K subunit. New drug candidates are routinely screened for their effects on the QT interval with tests focused on effects on the potassium channel. The findings of Lu et al. may require changes in how new drugs are tested. Many drugs, including some commonly used medications, can cause abnormal heart rhythms and sudden death, as manifest by a prolonged QT interval in the electrocardiogram. Cardiac arrhythmias caused by drug-induced long QT syndrome are thought to result mainly from reductions in the delayed rectifier potassium ion (K+) current IKr. Here, we report a mechanism for drug-induced QT prolongation that involves changes in multiple ion currents caused by a decrease in phosphoinositide 3-kinase (PI3K) signaling. Treatment of canine cardiac myocytes with inhibitors of tyrosine kinases or PI3Ks caused an increase in action potential duration that was reversed by intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate. The inhibitors decreased the delayed rectifier K+ currents IKr and IKs, the L-type calcium ion (Ca2+) current ICa,L, and the peak sodium ion (Na+) current INa and increased the persistent Na+ current INaP. Computer modeling of the canine ventricular action potential showed that the drug-induced change in any one current accounted for less than 50% of the increase in action potential duration. Mouse hearts lacking the PI3K p110α catalytic subunit exhibited a prolonged action potential and QT interval that were at least partly a result of an increase in INaP. These results indicate that down-regulation of PI3K signaling directly or indirectly via tyrosine kinase inhibition prolongs the QT interval by affecting multiple ion channels. This mechanism may explain why some tyrosine kinase inhibitors in clinical use are associated with increased risk of life-threatening arrhythmias.

Class IA PI3K p110β Subunit Promotes Autophagy through Rab5 Small GTPase in Response to Growth Factor Limitation

Autophagy is an evolutionarily conserved membrane trafficking process. Induction of autophagy in response to nutrient limitation or cellular stress occurs by similar mechanisms in organisms from yeast to mammals. Unlike yeast, metazoan cells rely more on growth factor signaling for a wide variety of cellular activities including nutrient uptake. How growth factor availability regulates autophagy is poorly understood. Here we show that, upon growth factor limitation, the p110β catalytic subunit of the class IA phosphoinositide 3-kinases (PI3Ks) dissociates from growth factor receptor complexes and increases its interaction with the small GTPase Rab5. This p110β-Rab5 association maintains Rab5 in its guanosine triphosphate (GTP)-bound state and enhances the Rab5-Vps34 interaction that promotes autophagy. p110β mutants that fail to interact with Rab5 are defective in autophagy promotion. Hence, in mammalian cells, p110β acts as a molecular sensor for growth factor availability and induces autophagy by activating a Rab5-mediated signaling cascade.

The class IA phosphatidylinositol 3-kinase p110-β subunit is a positive regulator of autophagy

p110-β associates with the Vps34–Vps15–Beclin 1–Atg14L complex and facilitates generation of PtdIns(3)P to promote autophagy.

Loss of Cardiac Phosphoinositide 3-Kinase p110α Results in Contractile Dysfunction

Background— Phosphoinositide 3-kinase (PI3K) p110&agr; plays a key role in insulin action and tumorigenesis. Myocyte contraction is initiated by an inward Ca2+ current (ICa,L) through the voltage-dependent L-type Ca2+ channel (LTCC). The aim of this study was to evaluate whether p110&agr; also controls cardiac contractility by regulating the LTCC. Methods and Results— Genetic ablation of p110&agr; (also known as Pik3ca), but not p110&bgr; (also known as Pik3cb), in cardiac myocytes of adult mice reduced ICa,L and blocked insulin signaling in the heart. p110&agr;-null myocytes had a reduced number of LTCCs on the cell surface and a contractile defect that decreased cardiac function in vivo. Similarly, pharmacological inhibition of p110&agr; decreased ICa,L and contractility in canine myocytes. Inhibition of p110&bgr; did not reduce ICa,L. Conclusions— PI3K p110&agr; but not p110&bgr; regulates the LTCC in cardiac myocytes. Decreased signaling to p110&agr; reduces the number of LTCCs on the cell surface and thus attenuates ICa,L and contractility.

Activated Gαq Inhibits p110α Phosphatidylinositol 3-Kinase and Akt

Some Gq-coupled receptors have been shown to antagonize growth factor activation of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt. We used a constitutively active Gαq(Q209L) mutant to explore the effects of Gαq activation on signaling through the PI3K/Akt pathway. Transient expression of Gαq(Q209L) in Rat-1 fibroblasts inhibited Akt activation induced by platelet-derived growth factor or insulin treatment. Expression of Gαq(Q209L) also attenuated Akt activation promoted by coexpression of constitutively active PI3K in human embryonic kidney 293 cells. Gαq(Q209L) had no effect on the activity of an Akt mutant in which the two regulatory phosphorylation sites were changed to acidic amino acids. Inducible expression of Gαq(Q209L) in a stably transfected 293 cell line caused a decrease in PI3K activity in p110α (but not p110β) immunoprecipitates. Receptor activation of Gαq also selectively inhibited PI3K activity in p110α immunoprecipitates. Active Gαq still inhibited PI3K/Akt in cells pretreated with the phospholipase C inhibitor U73122. Finally, Gαq(Q209L) co-immunoprecipitated with the p110α-p85α PI3K heterodimer from lysates of COS-7 cells expressing these proteins, and incubation of immunoprecipitated Gαq(Q209L) with purified recombinant p110α-p85α in vitro led to a decrease in PI3K activity. These results suggest that agonist binding to Gq-coupled receptors blocks Akt activation via the release of active Gαq subunits that inhibit PI3K. The inhibitory mechanism seems to be independent of phospholipase C activation and might involve an inhibitory interaction between Gαq and p110α PI3K.

Decreased L-type Ca2+ Current in Cardiac Myocytes of Type 1 Diabetic Akita Mice Due to Reduced Phosphatidylinositol 3-kinase Signaling

OBJECTIVE—Contraction of cardiac myocytes is initiated by Ca2+ entry through the voltage-dependent l-type Ca2+ channel (LTCC). Previous studies have shown that phosphatidylinositol (PI) 3-kinase signaling modulates LTCC function. Because PI 3-kinases are key mediators of insulin action, we investigated whether LTCC function is affected in diabetic animals due to reduced PI 3-kinase signaling. RESEARCH DESIGN AND METHODS—We used whole-cell patch clamping and biochemical assays to compare cardiac LTCC function and PI 3-kinase signaling in insulin-deficient diabetic mice heterozygous for the Ins2Akita mutation versus nondiabetic littermates. RESULTS—Diabetic mice had a cardiac contractility defect, reduced PI 3-kinase signaling in the heart, and decreased l-type Ca2+ current (ICa,L) density in myocytes compared with control nondiabetic littermates. The lower ICa,L density in myocytes from diabetic mice is due at least in part to reduced cell surface expression of the LTCC. ICa,L density in myocytes from diabetic mice was increased to control levels by insulin treatment or intracellular infusion of PI 3,4,5-trisphosphate [PI(3,4,5)P3]. This stimulatory effect was blocked by taxol, suggesting that PI(3,4,5)P3 stimulates microtubule-dependent trafficking of the LTCC to the cell surface. The voltage dependence of steady-state activation and inactivation of ICa,L was also shifted to more positive potentials in myocytes from diabetic versus nondiabetic animals. PI(3,4,5)P3 infusion eliminated only the difference in voltage dependence of steady-state inactivation of ICa,L. CONCLUSIONS—Decreased PI 3-kinase signaling in myocytes from type 1 diabetic mice leads to reduced Ca2+ entry through the LTCC, which might contribute to the negative effect of diabetes on cardiac contractility.

A Transgenic Mouse Model of Heart Failure Using Inducible Gαq

Receptors coupled to Gαq play a key role in the development of heart failure. Studies using genetically modified mice suggest that Gαq mediates a hypertrophic response in cardiac myocytes. Gαq signaling in these models is modified during early growth and development, whereas most heart failure in humans occurs after cardiac damage sustained during adulthood. To determine the phenotype of animals that express increased Gαq signaling only as adults, we generated transgenic mice that express a silent Gαq protein (GαqQ209L-hbER) in cardiac myocytes that can be activated by tamoxifen. Following drug treatment to activate Gαq Q209L-hbER, these mice rapidly develop a dilated cardiomyopathy and heart failure. This phenotype does not appear to involve myocyte hypertrophy but is associated with dephosphorylation of phospholamban (PLB), decreased sarcoplasmic reticulum Ca2+-ATPase activity, and a decrease in L-type Ca2+ current density. Changes in Ca2+ handling and decreased cardiac contractility are apparent 1 week after GαqQ209L-hbER activation. In contrast, transgenic mice that express an inducible Gαq mutant that cannot activate phospholipase Cβ (PLCβ) do not develop heart failure or changes in PLB phosphorylation, but do show decreased L-type Ca2+ current density. These results demonstrate that activation of Gαq in cardiac myocytes of adult mice causes a dilated cardiomyopathy that requires the activation of PLCβ. However, increased PLCβ signaling is not required for all of the Gαq-induced cardiac abnormalities.