PTEN phosphatase acts as a tumor suppressor by negatively regulating the phosphoinositide 3-kinase (PI3K) signaling pathway. It is unclear which downstream components of this pathway are necessary for oncogenic transformation. In this report we show that transformed cells of PTEN ؉/؊ mice have elevated levels of phosphorylated Akt and activated p70͞S6 kinase associated with an increase in proliferation. Pharmacological inactivation of mTOR͞ RAFT͞FRAP reduced neoplastic proliferation, tumor size, and p70͞S6 kinase activity, but did not affect the status of Akt. These data suggest that p70͞S6K and possibly other targets of mTOR contribute significantly to tumor development and that inhibition of these proteins may be therapeutic for cancer patients with deranged PI3K signaling.

Increased cardiac contractility during fight-or-flight response is caused by β-adrenergic augmentation of Ca V 1.2 channels 1-4. In transgenic murine hearts expressing fully PKA phosphorylation-site-deficient mutant Ca V 1.2 α 1C and β subunits, this regulation persists, implying involvement of extra-channel factors. Here, we identify the mechanism by which β-adrenergic agonists stimulate voltage-gated Ca 2+ channels. We expressed α 1C or β 2B subunits conjugated to ascorbate-peroxidase 5 in mouse hearts and used multiplexed, quantitative proteomics 6,7 to track hundreds of proteins in proximity of Ca V 1.2. We observed that the Ca 2+ channel inhibitor Rad 8,9 , a monomeric G-protein, is enriched in the Ca V 1.2 micro-environment but is depleted during β-adrenergic stimulation. PKA-catalyzed phosphorylation of specific Ser residues on Rad decreases its affinity for auxiliary β-Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

In the current study, we examined a panel of serially passaged glioblastoma xenografts, in the context of an intracranial tumor therapy response model, to identify associations between glioblastoma molecular characteristics and tumor sensitivity to the epidermal growth factor receptor (EGFR) kinase inhibitor erlotinib. From an initial evaluation of 11 distinct glioblastoma xenografts, two erlotinib-sensitive tumors were identified, each having amplified EGFR and expressing wild-type PTEN. One of these tumors expressed truncated EGFRvIII, whereas the other expressed full-length EGFR. Subsequent cDNA sequence analysis revealed the latter tumor as expressing an EGFR sequence variant with arginine, rather than leucine, at amino acid position 62; this was the only EGFR sequence variant identified among the 11 xenografts, other than the aforementioned vIII sequence variant. EGFR cDNAs were then examined from 12 more xenografts to determine whether additional missense sequence alterations were evident, and this analysis revealed one such case, expressing threonine, rather than alanine, at amino acid position 289 of the extracellular domain. This glioblastoma was also amplified for EGFR, but did not display significant erlotinib sensitivity, presumably due to its lacking PTEN expression. In total, our study identified two erlotinib-sensitive glioblastoma xenografts, with the common molecular characteristics shared by each being the expression of wild-type PTEN in combination with the expression of amplified and aberrant EGFR. [Mol Cancer Ther 2007;6(3):1167 -74]

BackgroundMisfolding of microtubule-associated protein tau (MAPT) within neurons into neurofibrillary tangles is an important pathological feature of Alzheimer’s disease (AD). Tau pathology correlates with cognitive decline in AD and follows a stereotypical anatomical course; several recent studies indicate that tau pathology spreads inter-neuronally via misfolded tau “seeds.” Previous research has focused on neurons as the source of these tau seeds. However, recent studies as well as the data contained herein suggest that microglia, the resident immune cells of the central nervous system, play a direct role in the spread of tau pathology.MethodsPrimary adult microglia were isolated from human AD cases and the rTg4510 tauopathy mouse model and used for analysis of gene expression, tau protein by Simoa technology, and quantification of tau seeding using a highly sensitive fluorescence resonance energy transfer (FRET) biosensing cell line for tau seeding and aggregation.ResultsHere, we show that microglia isolated from both human tauopathy and AD cases and the rTg4510 tauopathy mouse model stably contain tau seeds, despite not synthesizing any tau. Microglia releases these tau seeds in vitro into their conditioned media (CM). This suggests that microglia have taken up tau but are incapable of entirely neutralizing its seeding activity. Indeed, when in vitro microglia are given media containing tau seeds, they reduce (but do not eliminate) tau seeding. When microglia are treated with inflammagens such as lipopolysaccharide (LPS), interleukin-1β (IL1β), tumor necrosis factor α (TNFα), or amyloid-β, their ability to reduce tau seeding is unchanged and these factors do not induce seeding activity on their own.ConclusionsOverall, these data suggest that microglia have a complex role: they are capable of taking up and breaking down seed competent tau, but do so inefficiently and could therefore potentially play a role in the spread of tau pathology.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1309-z) contains supplementary material, which is available to authorized users.

The position and role of the unique N-terminal transmembrane (TM) helix, S0, in large-conductance, voltage-and calcium-activated potassium (BK) channels are undetermined. From the extents of intra-subunit, endogenous disulfi de bond formation between cysteines substituted for the residues just outside the membrane domain, we infer that the extracellular fl ank of S0 is surrounded on three sides by the extracellular fl anks of TM helices S1 and S2 and the four-residue extracellular loop between S3 and S4. Eight different double cysteine -substituted alphas, each with one cysteine in the S0 fl ank and one in the S3 -S4 loop, were at least 90% disulfi de cross-linked. Two of these alphas formed channels in which 90% cross-linking had no effect on the V 50 or on the activation and deactivation rate constants. This implies that the extracellular ends of S0, S3, and S4 are close in the resting state and move in concert during voltage sensor activation. The association of S0 with the gating charge bearing S3 and S4 could contribute to the considerably larger electrostatic energy required to activate the BK channel compared with typical voltage-gated potassium channels with six TM helices.

The cardiac-delayed rectifier K ؉ current (IKS) is carried by a complex of KCNQ1 (Q1) subunits, containing the voltage-sensor domains and the pore, and auxiliary KCNE1 (E1) subunits, required for the characteristic IKS voltage dependence and kinetics. To locate the transmembrane helix of E1 (E1-TM) relative to the Q1 TM helices (S1-S6), we mutated, one at a time, the first four residues flanking the extracellular ends of S1-S6 and E1-TM to Cys, coexpressed all combinations of Q1 and E1 Cys-substituted mutants in CHO cells, and determined the extents of spontaneous disulfide-bond formation. Cys-flanking E1-TM readily formed disulfides with Cysflanking S1 and S6, much less so with the S3-S4 linker, and not at all with S2 or S5. These results imply that the extracellular flank of the E1-TM is located between S1 and S6 on different subunits of Q1. The salient functional effects of selected cross-links were as follows. A disulfide from E1 K41C to S1 I145C strongly slowed deactivation, and one from E1 L42C to S6 V324C eliminated deactivation. Given that E1-TM is between S1 and S6 and that K41C and L42C are likely to point approximately oppositely, these two cross-links are likely to favor similar axial rotations of E1-TM. In the opposite orientation, a disulfide from E1 K41C to S6 V324C slightly slowed activation, and one from E1 L42C to S1 I145C slightly speeded deactivation. Thus, the first E1 orientation strongly favors the open state, while the approximately opposite orientation favors the closed state.arrhythmias ͉ cardiac repolarization ͉ electrophysiology ͉ atrial fibrillation ͉ S1T he slow, outwardly rectifying K ϩ current (I KS ) is one of two delayed rectifier K ϩ currents critical for repolarization of the heart, particularly during sympathetic nervous system stimulation (1, 2). The I KS channel is composed of four pore-forming KCNQ1 (Q1) subunits and two auxiliary KCNE1 (E1) subunits (3-5). Several human mutations in Q1 and E1 cause variants of long QT syndrome (6), short QT syndrome (7), or atrial fibrillation (8, 9).Although a tetramer of Q1 subunits alone forms a voltagegated channel, only Q1 and E1 together form a channel with the slow activation and deactivation kinetics and the minimal inactivation characteristics of I KS (10, 11). Furthermore, E1 is necessary for sympathetic modulation of I KS (12). How E1 exerts its effect on Q1 is not yet fully understood.There have been a number of conclusions about Q1-E1 interactions in the I KS channel, not all of which are compatible. There is evidence for (13) and against (14, 15) the contribution of E1 to the pore wall and its accessibility from the pore. There is also evidence that E1 interacts with the pore domain, although not necessarily exposed in the pore (16,17), that the E1 TM helix (E1-TM) interacts directly with Q1 S4 helix (18), that E1 modulates Q1 through its C terminus (19-21), and that E1 interacts with the cytoplasmic Q1 S4-S5 linker (22).More recently, a site of possible Q1-E1 interaction was suggested by the association of mutations in Q1 S...

No abstract

Large-conductance voltage and Ca 2 þ -activated potassium channels (BKCa) play a critical role in modulating contractile tone of smooth muscle, and neuronal processes. In most mammalian tissues, activation of b-adrenergic receptors and protein kinase A (PKA c ) increases BKCa channel activity, contributing to sympathetic nervous system/hormonal regulation of membrane excitability. Here we report the requirement of an association of the b2-adrenergic receptor (b2AR) with the pore forming a subunit of BKCa and an A-kinase-anchoring protein (AKAP79/150) for b2 agonist regulation. b2AR can simultaneously interact with both BKCa and L-type Ca 2 þ channels (Ca v 1.2) in vivo, which enables the assembly of a unique, highly localized signal transduction complex to mediate Ca 2 þ -and phosphorylation-dependent modulation of BKCa current. Our findings reveal a novel function for G protein-coupled receptors as a scaffold to couple two families of ion channels into a physical and functional signaling complex to modulate b-adrenergic regulation of membrane excitability.