Essential Role of the p110β Subunit of Phosphoinositide 3-OH Kinase in Male Fertility

Phosphoinositide 3-kinases (PI3K) are key molecular players in male fertility. However, the specific roles of different p110 PI3K catalytic subunits within the spermatogenic lineage have not been characterized so far. Herein, we report that male mice expressing a catalytically inactive p110β develop testicular hypotrophy and impaired spermatogenesis, leading to a phenotype of oligo-azoospermia and defective fertility. The examination of testes from p110β-defective tubules demonstrates a widespread loss in spermatogenic cells, due to defective proliferation and survival of pre- and postmeiotic cells. In particular, p110β is crucially needed in c-Kit–mediated spermatogonial expansion, as c-Kit–positive cells are lost in the adult testis and activation of Akt by SCF is blocked by a p110β inhibitor. These data establish that activation of the p110β PI3K isoform by c-Kit is required during spermatogenesis, thus opening the way to new treatments for c-Kit positive testicular cancers.     

Oncogenic Mutations of p110α Isoform of PI 3-Kinase Upregulate Its Protein Kinase Activity

In addition to lipid kinase activity, the class-I PI 3-kinases also function as protein kinases targeting regulatory autophosphorylation sites and exogenous substrates. The latter include a recently identified regulatory phosphorylation of the GM-CSF/IL-3 βc receptor contributing to survival of acute myeloid leukaemia cells. Previous studies suggested differences in the protein kinase activity of the 4 isoforms of class-I PI 3-kinase so we compared the ability of all class-I PI 3-kinases and 2 common oncogenic mutants to autophosphorylate, and to phosphorylate an intracellular fragment of the GM-CSF/IL-3 βc receptor (βic). We find p110α, p110β and p110γ all phosphorylate βic but p110δ is much less effective. The two most common oncogenic mutants of p110α, H1047R and E545K have stronger protein kinase activity than wildtype p110α, both in terms of autophosphorylation and towards βic. Importantly, the lipid kinase activity of the oncogenic mutants is still inhibited by autophosphorylation to a similar extent as wildtype p110α. Previous evidence indicates the protein kinase activity of p110α is Mn²⁺ dependent, casting doubt over its role in vivo. However, we show that the oncogenic mutants of p110α plus p110β and p110γ all display significant activity in the presence of Mg²⁺. Furthermore we demonstrate that some small molecule inhibitors of p110α lipid kinase activity (PIK-75 and A66) are equally effective against the protein kinase activity, but other inhibitors (e.g. wortmannin and TGX221) show different patterns of inhibition against the lipid and protein kinases activities. These findings have implications for the function of PI 3-kinase, especially in tumours carrying p110α mutations.     

Role of Phosphoinositide 3-OH Kinase p110β in Skeletal Myogenesis

Phosphoinositide 3-OH kinase (PI3K) regulates a number of developmental and physiologic processes in skeletal muscle; however, the contributions of individual PI3K p110 catalytic subunits to these processes are not well-defined. To address this question, we investigated the role of the 110-kDa PI3K catalytic subunit β (p110β) in myogenesis and metabolism. In C2C12 cells, pharmacological inhibition of p110β delayed differentiation. We next generated mice with conditional deletion of p110β in skeletal muscle (p110β muscle knockout [p110β-mKO] mice). While young p110β-mKO mice possessed a lower quadriceps mass and exhibited less strength than control littermates, no differences in muscle mass or strength were observed between genotypes in old mice. However, old p110β-mKO mice were less glucose tolerant than old control mice. Overexpression of p110β accelerated differentiation in C2C12 cells and primary human myoblasts through an Akt-dependent mechanism, while expression of kinase-inactive p110β had the opposite effect. p110β overexpression was unable to promote myoblast differentiation under conditions of p110α inhibition, but expression of p110α was able to promote differentiation under conditions of p110β inhibition. These findings reveal a role for p110β during myogenesis and demonstrate that long-term reduction of skeletal muscle p110β impairs whole-body glucose tolerance without affecting skeletal muscle size or strength in old mice.     

A New Dimension to Ras Function: A Novel Role for Nucleotide-Free Ras in Class II Phosphatidylinositol 3-Kinase Beta (PI3KC2β) Regulation

The intersectin 1 (ITSN1) scaffold stimulates Ras activation on endocytic vesicles without activating classic Ras effectors. The identification of Class II phosphatidylinositol 3-kinase beta, PI3KC2β, as an ITSN1 target on vesicles and the presence of a Ras binding domain (RBD) in PI3KC2β suggests a role for Ras in PI3KC2β activation. Here, we demonstrate that nucleotide-free Ras negatively regulates PI3KC2β activity. PI3KC2β preferentially interacts in vivo with dominant-negative (DN) Ras, which possesses a low affinity for nucleotides. PI3KC2β interaction with DN Ras is disrupted by switch 1 domain mutations in Ras as well as RBD mutations in PI3KC2β. Using purified proteins, we demonstrate that the PI3KC2β-RBD directly binds nucleotide-free Ras in vitro and that this interaction is not disrupted by nucleotide addition. Finally, nucleotide-free Ras but not GTP-loaded Ras inhibits PI3KC2β lipid kinase activity in vitro. Our findings indicate that PI3KC2β interacts with and is regulated by nucleotide-free Ras. These data suggest a novel role for nucleotide-free Ras in cell signaling in which PI3KC2β stabilizes nucleotide-free Ras and that interaction of Ras and PI3KC2β mutually inhibit one another.     

Role of connexin-43 in protective PI3K-Akt-GSK-3β signaling in cardiomyocytes

Sarcolemmal connexin-43 (Cx43) and mitochondrial Cx43 play distinct roles: formation of gap junctions and production of reactive oxygen species (ROS) for redox signaling. In this study, we examined the hypothesis that Cx43 contributes to activation of a major cytoprotective signal pathway, phosphoinositide 3-kinase (PI3K)-Akt-glycogen synthase kinase-3β (GSK-3β) signaling, in cardiomyocytes. A δ-opioid receptor agonist {[d-Ala(2),d-Leu(5)]enkephalin acetate (DADLE)}, endothelin-1 (ET-1), and insulin-like growth factor-1 (IGF-1) induced phosphorylation of Akt and GSK-3β in H9c2 cardiomyocytes. Reduction of Cx43 protein to 20% of the normal level by Cx43 small interfering RNA abolished phosphorylation of Akt and GSK-3β induced by DADLE or ET-1 but not that induced by IGF-1. DADLE and IGF-1 protected H9c2 cells from necrosis after treatment with H(2)O(2) or antimycin A. The protection by DADLE or ET-1, but not that by IGF-1, was lost by reduction of Cx43 protein expression. In contrast to Akt and GSK-3β, PKC-ε, ERK and p38 mitogen-activated protein kinase were phosphorylated by ET-1 in Cx43-knocked-down cells. Like diazoxide, an activator of the mitochondrial ATP-sensitive K(+) channel, DADLE and ET-1 induced significant ROS production in mitochondria, although such an effect was not observed for IGF-1. Cx43 knockdown did not attenuate the mitochondrial ROS production by DADLE or ET-1. Cx43 was coimmunoprecipitated with the β-subunit of G protein (Gβ), and knockdown of Gβ mimicked the effect of Cx43 knockdown on ET-1-induced phosphorylation of Akt and GSK-3β. These results suggest that Cx43 contributes to activation of class I(B) PI3K in PI3K-Akt-GSK-3β signaling possibly as a cofactor of Gβ in cardiomyocytes.     

Dual Role of α-Secretase Cleavage in the Regulation of γ-Secretase Activity for Amyloid Production

Processing of the amyloid precursor protein (APP) by β- and γ-secretases generates pathogenic β-amyloid (Aβ) peptides associated with Alzheimer disease (AD), whereas cleavage of APP by α-secretases precludes Aβ formation. Little is known about the role of α-secretase cleavage in γ-secretase regulation. Here, we show that α-secretase-cleaved APP C-terminal product (αCTF) functions as an inhibitor of γ-secretase. We demonstrate that the substrate inhibitory domain (ASID) within αCTF, which is bisected by the α-secretase cleavage site, contributes to this negative regulation because deleting or masking this domain turns αCTF into a better substrate for γ-secretase. Moreover, α-secretase cleavage can potentiate the inhibitory effect of ASID. Inhibition of γ-secretase activity by αCTF is observed in both in vitro and cellular systems. This work reveals an unforeseen role for α-secretase in generating an endogenous γ-secretase inhibitor that down-regulates the production of Aβ. Deregulation of this feedback mechanism may contribute to the pathogenesis of AD.     

Role of Novel Serine 316 Phosphorylation of the p65 Subunit of NF-κB in Differential Gene Regulation

Nuclear factor κB (NF-κB) is a central coordinator in immune and inflammatory responses. Constitutive NF-κB is often found in some types of cancers, contributing to oncogenesis and tumor progression. Therefore, knowing how NF-κB is regulated is important for its therapeutic control. Post-translational modification of the p65 subunit of NF-κB is a well known approach for its regulation. Here, we reported that in response to interleukin 1β, the p65 subunit of NF-κB is phosphorylated on the novel serine 316. Overexpression of S316A (serine 316 → alanine) mutant exhibited significantly reduced ability to activate NF-κB and decreased cell growth as compared with wtp65 (wild type p65). Moreover, conditioned media from cells expressing the S316A-p65 mutant had a considerably lower ability to induce NF-κB than that of wtp65. Our data suggested that phosphorylation of p65 on Ser-316 controls the activity and function of NF-κB. Importantly, we found that phosphorylation at the novel Ser-316 site and other two known phosphorylation sites, Ser-529 and Ser-536, either individually or cooperatively, regulated distinct groups of NF-κB-dependent genes, suggesting the unique role of each individual phosphorylation site on NF-κB-dependent gene regulation. Our novel findings provide an important piece of evidence regarding differential regulation of NF-κB-dependent genes through phosphorylation of different p65 serine residues, thus shedding light on novel mechanisms for the pathway-specific control of NF-κB. This knowledge is key to develop strategies for prevention and treatment of constitutive NF-κB-driven inflammatory diseases and cancers.     

Evidence for a role for the p110-α isoform of PI3K in skeletal function

Signaling through phosphatidylinositol-3 kinases (PI3K) regulates fundamental cellular processes such as survival and growth, and these lipid kinases are currently being investigated as therapeutic targets in several contexts. In skeletal tissue, experiments using pan-specific PI3K inhibitors have suggested that PI3K signaling influences both osteoclast and osteoblast function, but the contributions of specific PI3K isoforms to these effects have not been examined. In the current work, we assessed the effects of pharmacological inhibitors of the class Ia PI3Ks, α, β, and δ, on bone cell growth, differentiation and function in vitro. Each of the class Ia PI3K isoforms is expressed and functionally active in bone cells. No consistent effects of inhibitors of p110-β or p110-δ on bone cells were observed. Inhibitors of p110-α decreased osteoclastogenesis by 60-80% (p < 0.001 vs control) by direct actions on osteoclast precursors, and decreased the resorptive activity of mature osteoclasts by 60% (p < 0.01 vs control). The p110-α inhibitors also decreased the growth of osteoblastic and stromal cells (p < 0.001 vs control), and decreased differentiated osteoblast function by 30% (p < 0.05 vs control). These data suggest that signaling through the p110-α isoform of class Ia PI3Ks positively regulates the development and function of both osteoblasts and osteoclasts. Therapeutic agents that target this enzyme have the potential to significantly affect bone homeostasis, and evaluation of skeletal endpoints in clinical trials of such agents is warranted.     

The p101 subunit of PI3Kγ restores activation by Gβ mutants deficient in stimulating p110γ

G-protein-regulated PI3Kγ (phosphoinositide 3-kinase γ) plays a crucial role in inflammatory and allergic processes. PI3Kγ, a dimeric protein formed by the non-catalytic p101 and catalytic p110γ subunits, is stimulated by receptor-released Gβγ complexes. We have demonstrated previously that Gβγ stimulates both monomeric p110γ and dimeric p110γ/p101 lipid kinase activity in vitro. In order to identify the Gβ residues responsible for the Gβγ-PI3Kγ interaction, we examined Gβ1 mutants for their ability to stimulate lipid and protein kinase activities and to recruit PI3Kγ to lipid vesicles. Our findings revealed different interaction profiles of Gβ residues interacting with p110γ or p110γ/p101. Moreover, p101 was able to rescue the stimulatory activity of Gβ1 mutants incapable of modulating monomeric p110γ. In addition to the known adaptor function of p101, in the present paper we show a novel regulatory role of p101 in the activation of PI3Kγ.     

Both p110α and p110β isoforms of PI3K can modulate the impact of loss-of-function of the PTEN tumour suppressor

The PI3K (phosphoinositide 3-kinase) pathway is commonly activated in cancer as a consequence of inactivation of the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), a major negative regulator of PI3K signalling. In line with this important role of PTEN, mice that are heterozygous for a PTEN-null allele (PTEN+/? mice) spontaneously develop a variety of tumours in multiple organs. PTEN is a phosphatase with selectivity for PtdIns(3,4,5)P3, which is produced by the class I isoforms of PI3K (p110α, p110β, p110γ and p110δ). Previous studies indicated that PTEN-deficient cancer cell lines mainly depend on p110β, and that p110β, but not p110α, controls mouse prostate cancer development driven by PTEN loss. In the present study, we investigated whether the ubiquitously expressed p110α can also functionally interact with PTEN in cancer. Using genetic mouse models that mimic systemic administration of p110α- or p110β-selective inhibitors, we confirm that inactivation of p110β, but not p110α, inhibits prostate cancer development in PTEN+/? mice, but also find that p110α inactivation protects from glomerulonephritis, pheochromocytoma and thyroid cancer induced by PTEN loss. This indicates that p110α can modulate the impact of PTEN loss in disease and tumourigenesis. In primary and immortalized mouse fibroblast cell lines, both p110α and p110β controlled steady-state PtdIns(3,4,5)P3 levels and Akt signalling induced by heterozygous PTEN loss. In contrast, no correlation was found in primary mouse tissues between PtdIns(3,4,5)P3 levels, PI3K/PTEN genotype and cancer development. Taken together, our results from the present study show that inactivation of either p110α or p110β can counteract the impact of PTEN inactivation. The potential implications of these findings for PI3K-targeted therapy of cancer are discussed.     

Inhibition of Wnt and PI3K Signaling Modulates GSK-3β Activity and Induces Morphological Changes in Cortical Neurons: Role of Tau Phosphorylation

Glycogen synthase kinase GSK-3β has been identified as one of the major candidates mediating tau hyperphosphorylation at the same sites as those present in tau protein in brain from Alzheimer′s disease (AD) patients. However, the signal transduction pathways involved in the abnormal activation of GSK-3β, have not been completely elucidated. GSK-3β activity is repressed by the canonical Wnt signaling pathway, but it is also modulated through the PI3K/Akt route. Recent studies have suggested that Wnt signaling might be involved in the pathophysiology of AD. On the other hand, modulators of the PI3K pathway might be reduced during aging leading to a sustained activation of GSK-3β, which in turn would increase the risk of tau hyperphosphorylation. The role of Wnt and PI3K signaling inhibition on the extent of tau phosphorylation and neuronal morphology has not been completely elucidated. Thus, in the present investigation we analyzed the effects of different negative modulators of the Wnt and the PI3K pathways on GSK-3β activation and phosphorylation of tau at the PHF-1 epitope in cortical cultured neurons and hippocampal slices from adult rat brain. Changes in the microtubule network were also studied. We found that a variety of Wnt and PI3K inhibitors, significantly increased tau phosphorylation at the PHF-1 site, induced the disarrangement of the microtubule network and the accumulation of tau within cell bodies. These changes correlated with alterations in neuronal morphology. Special issue article in honor of Dr. Ricardo Tapia.     

Neuronal IP3 3-Kinase is an F-actin–bundling Protein: Role in Dendritic Targeting and Regulation of Spine Morphology

The actin microstructure in dendritic spines is involved in synaptic plasticity. Inositol trisphosphate 3-kinase A (ITPKA) terminates Ins(1,4,5)P₃ signals emanating from spines and also binds filamentous actin (F-actin) through its amino terminal region (amino acids 1-66, N66). Here we investigated how ITPKA, independent of its kinase activity, regulates dendritic spine F-actin microstructure. We show that the N66 region of the protein mediates F-actin bundling. An N66 fusion protein bundled F-actin in vitro, and the bundling involved N66 dimerization. By mutagenesis we identified a point mutation in a predicted helical region that eliminated both F-actin binding and bundling, rendering the enzyme cytosolic. A fusion protein containing a minimal helical region (amino acids 9-52, N9-52) bound F-actin in vitro and in cells, but had lower affinity. In hippocampal neurons, GFP-tagged N66 expression was highly polarized, with targeting of the enzyme predominantly to spines. By contrast, N9-52-GFP expression occurred in actin-rich structures in dendrites and growth cones. Expression of N66-GFP tripled the length of dendritic protrusions, induced longer dendritic spine necks, and induced polarized actin motility in time-lapse assays. These results suggest that, in addition to its ability to regulate intracellular Ca²⁺ via Ins(1,4,5)P₃ metabolism, ITPKA regulates structural plasticity.     

Lysophosphatidic acid-stimulated phosphorylation of PKD2 is mediated by PI3K p110β and PKCδ in myoblasts

Abstract

Context: G-protein coupled receptor (GPCR) signaling in skeletal muscle is incompletely understood; in particular, the signaling pathways that regulate GPCR-mediated signaling in skeletal muscle are only beginning to be established. Lysophosphatidic acid (LPA) is a GPCR agonist that has previously been shown to activate protein kinase D (PKD) in non-muscle cells; however, whether PKD is activated in response to LPA in skeletal muscle myoblasts, and the identities of signaling intermediates that regulate this activation, have not been defined. Objective: To determine whether PKD is activated in response to LPA administration in myoblasts, and to define the signaling pathways that mediate LPA-stimulated PKD phosphorylation. Methods: C2C12 myoblasts were treated with LPA and signaling pathways examined by means of Western immunoblotting and real-time PCR (RT-PCR). Pharmacological inhibition and RNA-interference were used to target specific molecules to determine their involvement in LPA-induced PKD phosphorylation. Results: Treatment of myoblasts with exogenous LPA revealed that PI3K p110β mediated PKD phosphorylation at Ser 748 and at Ser 916 through kinase-dependent and kinase-independent mechanisms. Loss of PKCδ, but not the loss of PKCα, prevented LPA-induced PKD phosphorylation. The PKD isoform responsive to LPA treatment was identified as PKD2. Conclusion: These results indicate that LPA-stimulated PKD2 phosphorylation requires PKCδ and non-catalytic actions of PI3K p110β, and provide new information with respect to GPCR-mediated signal transduction in myoblasts.