Class II phosphoinositide 3-kinases are downstream targets of activated polypeptide growth factor receptors

The class II phosphoinositide 3-kinases (PI3K) PI3K-C2alpha and PI3K-C2beta are two recently identified members of the large PI3K family. Both enzymes are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidylinositol 4-monophosphate as lipid substrates. Little is understood about how the catalytic activity of either enzyme is regulated in vivo. In this study, we demonstrate that PI3K-C2alpha and PI3K-C2beta represent two downstream targets of the activated epidermal growth factor (EGF) receptor in human carcinoma-derived A431 cells. Stimulation of quiescent cultures with EGF resulted in the rapid recruitment of both enzymes to a phosphotyrosine signaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3K-C2alpha and PI3K-C2beta immunoreactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca(2+) as an essential divalent cation in lipid kinase assays and since the catalytic activity of PI3K-C2alpha is refractory to the inhibitor wortmannin, these properties were used to confirm the recruitment of each PI3K isozyme to the activated EGF receptor complex. To examine this interaction in greater detail, PI3K-C2beta was chosen for further investigation. EGF and platelet-derived growth factor also stimulated the association of PI3K-C2beta with their respective receptors in other cells, including epithelial cells and fibroblasts. The use of EGF receptor mutants and phosphopeptides derived from the EGF receptor and Erb-B2 demonstrated that the interaction with recombinant PI3K-C2beta occurs through E(p)YL/I phosphotyrosine motifs. The N-terminal region of PI3K-C2beta was found to selectively interact with the EGF receptor in vitro, suggesting that it mediates the association of this PI3K with the receptor. However, the mechanism of this interaction remains unclear. We conclude that class II PI3K enzymes may contribute to the generation of 3' phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biological activity.     

Individual phosphoinositide 3-kinase C2alpha domain activities independently regulate clathrin function

Phosphoinositide 3-kinase C2alpha (PI3K-C2alpha) is a member of the class II PI-3 kinases, defined by the presence of a second C2 domain at their C termini. The cellular functions of the class II enzymes are incompletely understood, though they have been implicated in receptor activation pathways initiated by epidermal growth factor, insulin, and chemokines. PI3K-C2alpha was recently found to be localized to clathrin-coated membranes in the trans-Golgi network and at endocytic sites on the plasma membrane. Further, a specific binding site was identified for clathrin on the N terminus of PI3K-C2alpha, whose occupancy resulted in lipid kinase activation. Expression of PI3K-C2alpha in cells dramatically affected clathrin distribution and function in cells, leading to accumulation of intracellular clathrin-coated structures, which are visualized here at the ultrastructural level, and inhibition of clathrin-mediated transport from both the plasma membrane and the trans-Golgi network. In this study we have demonstrated that the isolated clathrin binding domain of PI3K-C2alpha can drive clathrin lattice assembly and that both it and the lipid kinase activity of the protein can independently modulate clathrin distribution and function when expressed in cells. Together, these results suggest that PI3K-C2alpha employs both protein-protein interaction and localized production of 3-phosphoinositides to affect clathrin dynamics at sites of membrane budding and targeting.     

The role of phosphoinositide 3-kinase C2alpha in insulin signaling

The members of the class II phosphoinositide 3-kinase (PI3K) family can be activated by several stimuli, indicating that these enzymes can regulate many intracellular processes. Nevertheless, to date, there has been no definitive identification of their in vivo product, their mechanism(s) of activation, or their precise intracellular roles. By metabolic labeling, we here identify phosphatidylinositol 3-phosphate as the sole in vivo product of the insulin-dependent activation of PI3K-C2alpha, confirming the emerging role of such a phosphoinositide in signaling. We demonstrate that activation of PI3K-C2alpha involves its recruitment to the plasma membrane and that activation is mediated by the GTPase TC10. This is the first report showing a membrane targeting-mediated mechanism of activation for PI3K-C2alpha and that a small GTP-binding protein can activate a class II PI3K isoform. We also demonstrate that PI3K-C2alpha contributes to maximal insulin-induced translocation of the glucose transporter GLUT4 to the plasma membrane and subsequent glucose uptake, definitely assessing the role of this enzyme in insulin signaling.     

Suppression of the alpha-isoform of class II phosphoinositide 3-kinase gene expression leads to apoptotic cell death

Phosphoinositide 3-kinases (PI3Ks) have known to be key enzymes activating intracellular signaling molecules when a number of growth factors bind to their cell surface receptors. PI3Ks are divided into three classes (I, II, and III) and enzymes of each class have different tissue-specificities and physiological functions. Class II PI3Ks consist of three isoforms (alpha,beta,gamma). Although the alpha-isoform (PI3K-C2alpha) is considered ubiquitous and preferentially activated by insulin rather than the beta-isoform, the physiological significance of PI3K-C2alpha is poorly understood. The present study aimed to determine whether PI3K-C2alpha is associated with the suppression of apoptotic cell death. Different sense- and antisense oligonucleotides (ODNs) were synthesized based on the sequence of C2 domain of PI3K-C2alpha gene. Transfection of CHO-IR cells with two different antisense ODNs clearly reduced the protein content as well as mRNA levels of PI3K-C2alpha whereas neither the nonspecific mock- nor sense ODNs affected. The decrease of PI3K-C2alpha gene expression was paralleled by cellular changes indicating apoptotic cell death such as nuclear condensation, formation of apoptotic bodies, and DNA fragmentation. PI3K-C2alpha mRNA levels were also reduced when cells were incubated in growth factor-deficient medium. Supplementing growth factors (serum or insulin) into medium lead to an increase of PI3K-C2alpha mRNA levels. This finding strongly suggests that PI3K-C2alpha is a crucial survival factor.     

Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells

Expression of the epithelial cell-specific heterotetrameric adaptor complex AP-1B is required for the polarized distribution of many membrane proteins to the basolateral surface of LLC-PK1 kidney cells. AP-1B is distinguished from the ubiquitously expressed AP-1A by exchange of its single 50-kD mu subunit, mu1A, being replaced by the closely related mu1B. Here we show that this substitution is sufficient to couple basolateral plasma membrane proteins, such as a low-density lipoprotein receptor (LDLR), to the AP-1B complex and to clathrin. The interaction between LDLR and AP-1B is likely to occur in the trans-Golgi network (TGN), as was suggested by the localization of functional, epitope-tagged mu1 by immunofluorescence and immunoelectron microscopy. Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles. Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes. We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.     

Functional cooperation of two independent targeting domains in syntaxin 6 is required for its efficient localization in the trans-golgi network of 3T3L1 adipocytes

To identify the targeting domains of syntaxin 6 responsible for its localization to the trans-Golgi network (TGN), we examined the subcellular distribution of enhanced green fluorescent protein (EGFP) epitope-tagged syntaxin 6/syntaxin 4 chimerae and syntaxin 6 truncation/deletion mutants in 3T3L1 adipocytes. Expression of EGFP-syntaxin 6 resulted in a perinuclear distribution identical to endogenous syntaxin 6 as determined both by confocal fluorescence microscopy and subcellular fractionation. Furthermore, both the endogenous and the expressed EGFP-syntaxin 6 fusion protein were localized to a brefeldin A-insensitive but okadaic acid-sensitive compartment characteristic of the TGN. In contrast, EGFP-syntaxin 6 constructs lacking the H2 domain were excluded from the TGN and were instead primarily localized to the plasma membrane. Although syntaxin 4 was localized to the plasma membrane, syntaxin 6/syntaxin 4 chimerae and syntaxin 6 truncations containing the H2 domain of syntaxin 6 were predominantly directed to the TGN. Importantly, the syntaxin 6 H2 domain fused to the transmembrane domain of syntaxin 4 was also localized to the TGN, demonstrating that the H2 domain was sufficient to confer TGN localization. In addition to the H2 domain, a tyrosine-based plasma membrane internalization signal (YGRL) was identified between the H1 and H2 domains of syntaxin 6. Deletion of this sequence resulted in the accumulation of the EGFP-syntaxin 6 reporter construct at the plasma membrane. Together, these data demonstrate that syntaxin 6 utilizes two distinct domains to drive its specific subcellular localization to the TGN.     

Ca2+-regulated pool of phosphatidylinositol-3-phosphate produced by phosphatidylinositol 3-kinase C2alpha on neurosecretory vesicles

Phosphatidylinositol-3-phosphate [PtdIns(3)P] is a key player in early endosomal trafficking and is mainly produced by class III phosphatidylinositol 3-kinase (PI3K). In neurosecretory cells, class II PI3K-C2alpha and its lipid product PtdIns(3)P have recently been shown to play a critical role during neuroexocytosis, suggesting that two distinct pools of PtdIns(3)P might coexist in these cells. However, the precise characterization of this additional pool of PtdIns(3)P remains to be established. Using a selective PtdIns(3)P probe, we have identified a novel PtdIns(3)P-positive pool localized on secretory vesicles, sensitive to PI3K-C2alpha knockdown and relatively resistant to wortmannin treatment. In neurosecretory cells, stimulation of exocytosis promoted a transient albeit large increase in PtdIns(3)P production localized on secretory vesicles sensitive to PI3K-C2alpha knockdown and expression of PI3K-C2alpha catalytically inactive mutant. Using purified chromaffin granules, we found that PtdIns(3)P production is controlled by Ca(2+). We confirmed that PtdIns(3)P production from recombinantly expressed PI3K-C2alpha is indeed regulated by Ca(2+). We provide evidence that a dynamic pool of PtdIns(3)P synthesized by PI3K-C2alpha occurs on secretory vesicles in neurosecretory cells, demonstrating that the activity of a member of the PI3K family is regulated by Ca(2+) in vitro and in living neurosecretory cells.     

The class II phosphoinositide 3-kinase C2alpha is activated by clathrin and regulates clathrin-mediated membrane trafficking

Phosphoinositides play key regulatory roles in vesicular transport pathways in eukaryotic cells. Clathrin-mediated membrane trafficking has been shown to require phosphoinositides, but little is known about the enzyme(s) responsible for their formation. Here we report that clathrin functions as an adaptor for the class II PI 3-kinase C2alpha (PI3K-C2alpha), binding to its N-terminal region and stimulating its catalytic activity, especially toward phosphorylated inositide substrates. Further, we show that endogenous PI3K-C2alpha is localized in coated pits and that exogenous expression affects clathrin-mediated endocytosis and sorting in the trans-Golgi network. These findings provide a mechanistic basis for localized inositide generation at sites of clathrin-coated bud formation, which, with recruitment of inositide binding proteins and subsequent synaptojanin-mediated phosphoinositide hydrolysis, may regulate coated vesicle formation and uncoating.     

Recruitment of the class II phosphoinositide 3-kinase C2beta to the epidermal growth factor receptor: role of Grb2

Previously we demonstrated that the class II phosphoinositide 3-kinase C2beta (PI3K-C2beta) is rapidly recruited to a phosphotyrosine signaling complex containing the activated receptor for epidermal growth factor (EGF). Although this association was shown to be dependent upon specific phosphotyrosine residues present on the EGF receptor, the underlying mechanism remained unclear. In this study the interaction between PI3K-C2beta and the EGF receptor is competitively attenuated by synthetic peptides derived from each of three proline-rich motifs present within the N-terminal region of the PI3K. Further, a series of N-terminal PI3K-C2beta fragments, truncated prior to each proline-rich region, bound the receptor with decreased efficiency. A single proline-rich region was unable to mediate receptor association. Finally, an equivalent N-terminal fragment of PI3K-C2alpha that lacks similar proline-rich motifs was unable to affinity purify the activated EGF receptor from cell lysates. Since these findings revealed that the interaction between the EGF receptor and PI3K-C2beta is indirect, we sought to identify an adaptor molecule that could mediate their association. In addition to the EGF receptor, PI3K-C2beta(2-298) also isolated both Shc and Grb2 from A431 cell lysates. Recombinant Grb2 directly bound PI3K-C2beta in vitro, and this effect was reproduced using either SH3 domain expressed as a glutathione S-transferase (GST) fusion. Interaction with Grb2 dramatically increased the catalytic activity of this PI3K. The relevance of this association was confirmed when PI3K-C2beta was isolated by coimmunoprecipitation with anti-Grb2 antibody from numerous cell lines. Using immobilized, phosphorylated EGF receptor, recombinant PI3K-C2beta was only purified in the presence of Grb2. We conclude that proline-rich motifs within the N terminus of PI3K-C2beta mediate the association of this enzyme with activated EGF receptor and that this interaction involves the Grb2 adaptor.     

Insulin activates the alpha isoform of class II phosphoinositide 3-kinase.

The novel class II phosphoinositide (PI) 3-kinases are characterized by the presence of a C-terminal C2 domain, but little is known about their regulation. We find insulin causes a rapid 2-3-fold increase in the activity of PI 3-kinase C2alpha (PI3K-C2alpha) in CHO-IR cells, 3T3-L1 adipocytes, and fully differentiated L5L6 myotubes. No insulin-induced activation of PI3K-C2alpha was observed in cell types known to have low responsiveness to insulin including HEK 293 cells, 3T3-L1 preadipocytes, and undifferentiated L5L6 myoblasts. The mechanism of activation of PI3K-C2alpha by insulin differs from that of class Ia PI 3-kinases in that insulin stimulation did not cause PI3K-C2alpha to associate with IRS-1 or insulin receptor. PI3K-C2alpha existed as a doublet, and insulin stimulation caused a redistribution from the lower molecular weight band to the higher molecular weight band, suggesting phosphorylation-induced bandshift. Consistent with this, in vitro phosphatase treatment reduced the intensity of the upper band back to that seen in unstimulated cells. This suggests that insulin-induced phosphorylation could play a role in regulation of the activity of PI3K-C2alpha. The finding that insulin activates PI3K-C2alpha in cell types known to possess a wide range of responses to insulin suggests that PI3K-C2alpha is a novel component of insulin-stimulated signaling cascades.     

Phosphatidylinositol 3-kinase C2alpha is essential for ATP-dependent priming of neurosecretory granule exocytosis

Neurotransmitter release and hormonal secretion are highly regulated processes culminating in the calcium-dependent fusion of secretory vesicles with the plasma membrane. Here, we have identified a role for phosphatidylinositol 3-kinase C2alpha (PI3K-C2alpha) and its main catalytic product, PtdIns3P, in regulated exocytosis. In neuroendocrine cells, PI3K-C2alpha is present on a subpopulation of mature secretory granules. Impairment of PI3K-C2alpha function specifically inhibits the ATP-dependent priming phase of exocytosis. Overexpression of wild-type PI3K-C2alpha enhanced secretion, whereas transfection of PC12 cells with a catalytically inactive PI3K-C2alpha mutant or a 2xFYVE domain sequestering PtdIns3P abolished secretion. Based on these results, we propose that production of PtdIns3P by PI3K-C2alpha is required for acquisition of fusion competence in neurosecretion.     

P4-ATPase requirement for AP-1/clathrin function in protein transport from the trans-Golgi network and early endosomes

Drs2p is a resident type 4 P-type ATPase (P4-ATPase) and potential phospholipid translocase of the trans-Golgi network (TGN) where it has been implicated in clathrin function. However, precise protein transport pathways requiring Drs2p and how it contributes to clathrin-coated vesicle budding remain unclear. Here we show a functional codependence between Drs2p and the AP-1 clathrin adaptor in protein sorting at the TGN and early endosomes of Saccharomyces cerevisiae. Genetic criteria indicate that Drs2p and AP-1 operate in the same pathway and that AP-1 requires Drs2p for function. In addition, we show that loss of AP-1 markedly increases Drs2p trafficking to the plasma membrane, but does not perturb retrieval of Drs2p from the early endosome back to the TGN. Thus AP-1 is required at the TGN to sort Drs2p out of the exocytic pathway, presumably for delivery to the early endosome. Moreover, a conditional allele that inactivates Drs2p phospholipid translocase (flippase) activity disrupts its own transport in this AP-1 pathway. Drs2p physically interacts with AP-1; however, AP-1 and clathrin are both recruited normally to the TGN in drs2Delta cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN.     

Phosphatidylinositol-4-kinase type II alpha is a component of adaptor protein-3-derived vesicles

A membrane fraction enriched in vesicles containing the adaptor protein (AP) -3 cargo zinc transporter 3 was generated from PC12 cells and was used to identify new components of these organelles by mass spectrometry. Proteins prominently represented in the fraction included AP-3 subunits, synaptic vesicle proteins, and lysosomal proteins known to be sorted in an AP-3-dependent way or to interact genetically with AP-3. A protein enriched in this fraction was phosphatidylinositol-4-kinase type IIalpha (PI4KIIalpha). Biochemical, pharmacological, and morphological analyses supported the presence of PI4KIIalpha in AP-3-positive organelles. Furthermore, the subcellular localization of PI4KIIalpha was altered in cells from AP-3-deficient mocha mutant mice. The PI4KIIalpha normally present both in perinuclear and peripheral organelles was substantially decreased in the peripheral membranes of AP-3-deficient mocha fibroblasts. In addition, as is the case for other proteins sorted in an AP-3-dependent way, PI4KIIalpha content was strongly reduced in nerve terminals of mocha hippocampal mossy fibers. The functional relationship between AP-3 and PI4KIIalpha was further explored by PI4KIIalpha knockdown experiments. Reduction of the cellular content of PI4KIIalpha strongly decreased the punctate distribution of AP-3 observed in PC12 cells. These results indicate that PI4KIIalpha is present on AP-3 organelles where it regulates AP-3 function.     

Phosphoinositide 3-kinase C2alpha links clathrin to microtubule-dependent movement

Phosphoinositide 3-kinase C2alpha (PI3K-C2alpha) is a type II PI-3-kinase that has been implicated in several important membrane transport and signaling processes. We previously found that overexpression of PI3K-C2alpha inhibits clathrin-mediated membrane trafficking and induces proliferation of novel clathrin-coated structures within the cytoplasm. Using fluorescently tagged fusions of PI3K-C2alpha and clathrin, we explored the behavior of these structures in intact cells. Both proteins are present in the structures, and using rapid image acquisition and fluorescence photoactivation probes, we find that they exhibit localized, rapid mobility (5-20 microm/s). The movement is micro-tubule-based as revealed by use of inhibitors, and PI3K-C2alpha accumulates on microtubules rapidly and reversibly following cytoplasmic acidification, which also blocks movement. Dynactin mediates the movement of these clathrin-PI3K-C2alpha structures, since disruption of dynactin function by overexpression of its p50 subunit also inhibits movement. Finally, immunoprecipitation experiments reveal an interaction between endogenous PI3K-C2alpha and dynactin subunits. Together, these results reveal a molecular linkage between PI3K-C2alpha and the microtubule motor machinery, with implications for membrane trafficking in intact cells.     

The α-isoform of class II phosphoinositide 3-kinase is more effectively activated by insulin receptors than IGF receptors, and activation requires receptor NPEY motifs

Little is known about the physiological role and mechanism of activation of class II phosphoinositide 3-kinases (PI3Ks), although it has been shown that the PI3K-C2alpha isoform is activated by insulin. Using chimaeric receptor constructs which can be activated independently of endogenous receptors in transfected cells, we found that PI3K-C2alpha activity was stimulated to a greater extent by insulin receptors than IGF receptors in 3T3-L1 adipocytes. Activation of PI3K-C2alpha required an intact NPEY motif in the receptor juxtamembrane domain. We conclude that PI3K-C2alpha is a candidate for participation in insulin-specific intracellular signalling.     

The N-terminus of phosphoinositide 3-kinase-C2beta regulates lipid kinase activity and binding to clathrin

The class II phosphoinositide 3-kinase (PI3K)-C2beta is recruited to polypeptide growth factor receptors following ligand stimulation. In contrast to the class I A p85/p110 heterodimers, this interaction is dependent upon proline residues present within the N-terminal sequence of the 3-phosphoinositide kinase. However, the mechanism by which PI3K-C2beta catalytic activity is regulated currently remains unknown. In many tumours, increased expression of ErbB receptors confers a poor prognosis. We demonstrate that increased expression of EGFR enhanced its association with PI3K-C2beta following stimulation with EGF. Deletion of the first proline rich region within the N-terminus precluded recruitment of PI3K-C2beta to activated EGFR. Although deletion of the first proline rich motif also rendered the enzyme catalytically inactive, further deletions (residues 1-148 and 1-261) that removed the second and third proline rich motifs increased kinase activity. These data confirm a regulatory role for the N-terminus of class II PI3K enzymes suggesting that catalytic activity is regulated by factors that associate with this region during recruitment to activated growth factor receptors. Using an N-terminal PI3K-C2beta-GST fusion protein, clathrin heavy chain was affinity purified from A431 cell lysates. Association of PI3K-C2beta with clathrin was confirmed by co-immunoprecipitation from cell lysates while intracellular co-localisation of PI3K-C2beta and clathrin was confirmed by confocal microscopy. Our findings demonstrate for the first time that the PI3K-C2beta isoform associates with clathrin and thus provides a link between receptor mediated intracellular signalling and clathrin coated vesicle transport.     

The phosphatidylinositol 3-kinase inhibitor wortmannin alters the metabolism of the Alzheimer's amyloid precursor protein

One of the hallmarks of Alzheimer's disease is the accumulation of senile plaques in brain, extracellular lesions comprised mostly of aggregates of the amyloid beta-peptide (Abeta). Abeta is proteolytically derived from the Alzheimer's amyloid precursor protein (APP). The generation of Abeta and nonamyloidogenic derivatives of APP involves utilization of alternative processing pathways and multiple subcellular compartments. To improve our understanding of the regulation of APP processing, we investigated the effects of wortmannin, a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, on APP processing. PI3-kinases form a multifaceted family of enzymes that represent converging points for multiple signal transduction pathways and also act as key regulators of vesicular trafficking. In N2a neuroblastoma cells expressing either wild-type APP or the "Swedish" familial Alzheimer's disease-associated mutant variant of APP, wortmannin treatment resulted in decreased release of both Abeta and soluble APPalpha. In parallel, full-length APP and both processed derivatives accumulated inside the cells. These effects were not present at nanomolar concentrations of wortmannin, but only at micromolar concentrations, implying the possible involvement of a recently described trans-Golgi network (TGN)-associated PI3-kinase that is resistant to nanomolar concentrations of the inhibitor, but sensitive to micromolar concentrations. All effects were reversible when the drug was removed from the cell culture medium. Given the suspected site of action of this novel PI3-kinase activity at the TGN, it is tempting to speculate that the unexpected increase in the levels of both intracellular soluble APPalpha and intracellular Abeta might be due to wortmannin-induced covesiculation of APP together with its respective secretase enzymes within the TGN, leading to the execution of alpha-, beta-, and gamma-secretase reactions.     

Overexpression of phosphoinositide-3-kinase class II alpha enhances mesenchymal stem cell survival in infarcted myocardium

The efficacy of mesenchymal stem cell (MSC) therapy for myocardial regeneration is limited by the poor survival of stem cells after transplantation into the infarcted heart. To improve the cell survival of MSCs in the infarcted heart, MSCs were genetically engineered to overexpress phosphoinositide-3-kinase class II alpha (PI3K-C2α). PI3K-C2α overexpression increased PI3K expression and the cell viability of MSCs. Furthermore, levels of survival-related phosphorylation were elevated in PI3K-C2α-MSCs. But, the level of apoptotic proteins downregulated and the number of PI-positive cells decreased in PI3K-C2α-MSCs compared to hypoxic MSCs. Nine rats per group had 1 × 10⁶ cells (20 μl PBS) transplanted after myocardial infarction. One week after transplantation, infarct size and area of fibrosis were reduced in the PI3K-C2α-MSC-transplanted group. The number of TUNEL positive cells declined, while the mean microvessel count per field was higher in the PI3K-C2α-MSC group than the MSC-injected group. Heart function was improved in the PI3K-C2α-MSCs group as assessed using a Millar catheter at 3 weeks after transplantation. These findings suggest that overexpression of PI3K-C2α in MSCs can assist cell survival and enhance myocardial regeneration.