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1.
Oncotarget ; 7(35): 56170-56182, 2016 Aug 30.
Article in English | MEDLINE | ID: mdl-27472465

ABSTRACT

Understanding the complex interaction between growth factor and steroid hormone signaling pathways in breast cancer is key to identifying suitable therapeutic strategies to avoid progression and therapy resistance. The interaction between these two pathways is of paramount importance for the development of endocrine resistance. Nevertheless, the molecular mechanisms behind their crosstalk are still largely obscure. We previously reported that Memo is a small redox-active protein that controls heregulin-mediated migration of breast cancer cells. Here we report that Memo sits at the intersection between heregulin and estrogen signaling, and that Memo controls Estrogen Receptor alpha (ERα) sub-cellular localization, phosphorylation, and function downstream of heregulin and estrogen in breast cancer cells. Memo facilitates ERα and c-Src interaction, ERα Y537 phosphorylation, and has the ability to control ERα extra-nuclear localization. Thus, we identify Memo as an important key mediator between the heregulin and estrogen signaling pathways, which affects both breast cancer cell migration and proliferation.


Subject(s)
Breast Neoplasms/metabolism , Estrogen Receptor alpha/metabolism , Estrogens/metabolism , Nonheme Iron Proteins/metabolism , src-Family Kinases/metabolism , Antineoplastic Agents, Hormonal/pharmacology , Antineoplastic Agents, Hormonal/therapeutic use , Breast Neoplasms/drug therapy , CSK Tyrosine-Protein Kinase , Cell Movement , Cell Nucleus/metabolism , Disease Progression , Drug Resistance, Neoplasm , Female , Gene Knockdown Techniques , Humans , Intracellular Signaling Peptides and Proteins , MCF-7 Cells , Microscopy, Fluorescence , Neuregulin-1/metabolism , Nonheme Iron Proteins/genetics , Phosphorylation , Signal Transduction
2.
J Biol Chem ; 290(29): 17999-18008, 2015 Jul 17.
Article in English | MEDLINE | ID: mdl-26055712

ABSTRACT

Connecdenn 1/2 are DENN (differentially expressed in normal and neoplastic cells) domain-bearing proteins that function as GEFs (guanine nucleotide exchange factors) for the small GTPase Rab35. Disruption of connecdenn/Rab35 function leads to defects in the recycling of multiple cargo proteins from endosomes with altered cell function, yet the regulation of connecdenn GEF activity is unexplored. We now demonstrate that connecdenn 1/2 are autoinhibited such that the purified, full-length proteins have significantly less Rab35 binding and GEF activity than the isolated DENN domain. Both proteins are phosphorylated with prominent phosphorylation sites between residues 500 and 600 of connecdenn 1. A large scale proteomics screen revealed that connecdenn 1 is phosphorylated at residues Ser-536 and Ser-538 in an Akt-dependent manner in response to insulin stimulation of adipocytes. Interestingly, we find that an Akt inhibitor reduces connecdenn 1 interaction with Rab35 after insulin treatment of adipocytes. Remarkably, a peptide flanking Ser-536/Ser-538 binds the DENN domain of connecdenn 1, whereas a phosphomimetic peptide does not. Moreover, connecdenn 1 interacts with 14-3-3 proteins, and this interaction is also disrupted by Akt inhibition and by mutation of Ser-536/Ser-538. We propose that Akt phosphorylation of connecdenn 1 downstream of insulin activation regulates connecdenn 1 function through an intramolecular interaction.


Subject(s)
Death Domain Receptor Signaling Adaptor Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , rab GTP-Binding Proteins/metabolism , 14-3-3 Proteins/metabolism , 3T3-L1 Cells , Animals , Death Domain Receptor Signaling Adaptor Proteins/chemistry , Guanine Nucleotide Exchange Factors/chemistry , HEK293 Cells , Humans , Insulin/metabolism , Mice , Phosphorylation , Protein Interaction Maps , Protein Structure, Tertiary , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction
3.
J Mol Endocrinol ; 53(1): 117-30, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24859970

ABSTRACT

cAMP-response element-binding protein (CREB) is required for the induction of adipogenic transcription factors such as CCAAT/enhancer-binding proteins (C/EBPs). Interestingly, it is known from studies in other tissues that LKB1 and its substrates AMP-activated protein kinase (AMPK) and salt-inducible kinases (SIKs) negatively regulate gene expression by phosphorylating the CREB co-activator CRTC2 and class IIa histone deacetylases (HDACs), which results in their exclusion from the nucleus where they co-activate or inhibit their targets. In this study, we show that AMPK/SIK signalling is acutely attenuated during adipogenic differentiation of 3T3-L1 preadipocytes, which coincides with the dephosphorylation and nuclear translocation of CRTC2 and HDAC4. When subjected to differentiation, 3T3-L1 preadipocytes in which the expression of LKB1 was stably reduced using shRNA (Lkb1-shRNA), as well as Lkb1-knockout mouse embryonic fibroblasts (Lkb1(-/-) MEFs), differentiated more readily into adipocyte-like cells and accumulated more triglycerides compared with scrambled-shRNA-expressing 3T3-L1 cells or Wt MEFs. In addition, the phosphorylation of CRTC2 and HDAC4 was reduced, and the mRNA expression of adipogenic transcription factors Cebpa, peroxisome proliferator-activated receptor γ (Pparg) and adipocyte-specific proteins such as hormone-sensitive lipase (HSL), fatty acid synthase (FAS), aP2, GLUT4 and adiponectin was increased in the absence of LKB1. The mRNA and protein expression of Ddit3/CHOP10, a dominant-negative member of the C/EBP family, was reduced in Lkb1-shRNA-expressing cells, providing a potential mechanism for the up-regulation of Pparg and Cebpa expression. These results support the hypothesis that LKB1 signalling keeps preadipocytes in their non-differentiated form.


Subject(s)
Adipogenesis/physiology , Protein Serine-Threonine Kinases/metabolism , 3T3-L1 Cells , AMP-Activated Protein Kinases/metabolism , Active Transport, Cell Nucleus , Adipocytes/cytology , Adipocytes/metabolism , Adipogenesis/genetics , Animals , Cell Differentiation , Cells, Cultured , Gene Knockout Techniques , Histone Deacetylases/metabolism , Mice , Models, Biological , Phosphorylation , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/genetics , RNA, Small Interfering/genetics , Signal Transduction , Transcription Factors/metabolism
4.
Radiol Manage ; 33(6): 22-6; quiz 27-8, 2011.
Article in English | MEDLINE | ID: mdl-22235736

ABSTRACT

The primary goals of the Patient Protection and Affordable Care Act are to expand insurance coverage through an individual mandate, and to reduce growing healthcare costs through new risk-based payment models and the formation of ACOs. With the high cost of exams and steady growth through the last decade, imaging appears to be a prime target for savings under accountable care. Given that some of the reform payment models are set to begin as early as next year, and private payers are increasingly instituting similar risk-based payment models in their plans, it is critical for imaging leaders to understand how these models will affect their growth strategy and prepare accordingly. A thorough analysis of the various payment models, considering all possible targets for cost savings, is required to accurately determine the timing and impact for imaging.


Subject(s)
Accountable Care Organizations , Diagnostic Imaging/economics , Patient Protection and Affordable Care Act , Education, Continuing , Reimbursement Mechanisms , United States
5.
Sci Signal ; 1(41): ra9, 2008 Oct 14.
Article in English | MEDLINE | ID: mdl-18922788

ABSTRACT

The gamma-aminobutyric acid (GABA) type A receptor (GABA(A)R) is the major inhibitory neurotransmitter receptor in the brain. Its multiple subunits show regional, developmental, and disease-related plasticity of expression; however, the regulatory networks controlling GABA(A)R subunit expression remain poorly understood. We report that the seizure-induced decrease in GABA(A)R alpha1 subunit expression associated with epilepsy is mediated by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway regulated by brain-derived neurotrophic factor (BDNF). BDNF- and seizure-dependent phosphorylation of STAT3 cause the adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB) family member ICER (inducible cAMP early repressor) to bind with phosphorylated CREB at the Gabra1:CRE site. JAK/STAT pathway inhibition prevents the seizure-induced decrease in GABA(A)R alpha1 abundance in vivo and, given that BDNF is known to increase the abundance of GABA(A)R alpha4 in a JAK/STAT-independent manner, indicates that BDNF acts through at least two distinct pathways to influence GABA(A)R-dependent synaptic inhibition.


Subject(s)
Brain-Derived Neurotrophic Factor/metabolism , Gene Expression Regulation , Janus Kinases/metabolism , Receptors, GABA-A/biosynthesis , STAT3 Transcription Factor/metabolism , Animals , Brain-Derived Neurotrophic Factor/genetics , CREB-Binding Protein/genetics , CREB-Binding Protein/metabolism , Cyclic AMP Response Element Modulator/genetics , Cyclic AMP Response Element Modulator/metabolism , Epilepsy/genetics , Epilepsy/metabolism , Humans , Janus Kinases/genetics , Male , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/genetics , STAT3 Transcription Factor/genetics , Signal Transduction/genetics , Synaptic Membranes/genetics , Synaptic Membranes/metabolism
6.
J Biol Chem ; 283(14): 9328-40, 2008 Apr 04.
Article in English | MEDLINE | ID: mdl-18180303

ABSTRACT

The regulated expression of type A gamma-aminobutyric acid (GABA) receptor (GABA(A)R) subunit genes plays a critical role in neuronal maturation and synaptogenesis. It is also associated with a variety of neurological diseases. Changes in GABA(A) receptor alpha1 subunit gene (GABRA1) expression have been reported in animal models of epilepsy, alcohol abuse, withdrawal, and stress. Understanding the genetic mechanism behind such changes in alpha subunit expression will lead to a better understanding of the role that signal transduction plays in control over GABA(A)R function and brings with it the promise of providing new therapeutic tools for the prevention or cure of a variety of neurological disorders. Here we show that activation of protein kinase C increases alpha1 subunit levels via phosphorylation of CREB (pCREB) that is bound to the GABRA1 promoter (GABRA1p). In contrast, activation of protein kinase A decreases levels of alpha1 even in the presence of pCREB. Decrease of alpha1 is dependent upon the inducible cAMP early repressor (ICER) as directly demonstrated by ICER-induced down-regulation of endogenous alpha1-containing GABA(A)Rs at the cell surface of cortical neurons. Taken together with the fact that there are less alpha1gamma2-containing GABA(A)Rs in neurons after protein kinase A stimulation and that activation of endogenous dopamine receptors down-regulates alpha1 subunit mRNA levels subsequent to induction of ICER, our studies identify a transcriptional mechanism for regulating the cell surface expression of alpha1-containing GABA(A)Rs that is dependent upon the formation of CREB heterodimers.


Subject(s)
CREB-Binding Protein/metabolism , Cerebral Cortex/metabolism , Cyclic AMP Response Element Modulator/metabolism , Neurons/metabolism , Receptors, GABA-A/biosynthesis , Response Elements , Transcription, Genetic , Alcoholism/metabolism , Alcoholism/pathology , Animals , CREB-Binding Protein/genetics , Cells, Cultured , Cerebral Cortex/pathology , Cyclic AMP-Dependent Protein Kinases/metabolism , Disease Models, Animal , Down-Regulation , Enzyme Activation , Epilepsy/metabolism , Epilepsy/pathology , Neurons/pathology , Protein Kinase C/metabolism , RNA, Messenger/biosynthesis , Rats , Synaptosomes/metabolism
7.
J Neurosci ; 26(44): 11342-6, 2006 Nov 01.
Article in English | MEDLINE | ID: mdl-17079662

ABSTRACT

Differential expression of GABA(A) receptor (GABR) subunits has been demonstrated in hippocampus from patients and animals with temporal lobe epilepsy (TLE), but whether these changes are important for epileptogenesis remains unknown. Previous studies in the adult rat pilocarpine model of TLE found reduced expression of GABR alpha1 subunits and increased expression of alpha4 subunits in dentate gyrus (DG) of epileptic rats compared with controls. To investigate whether this altered subunit expression is a critical determinant of spontaneous seizure development, we used adeno-associated virus type 2 containing the alpha4 subunit gene (GABRA4) promoter to drive transgene expression in DG after status epilepticus (SE). This novel use of a condition-dependent promoter upregulated after SE successfully increased expression of GABR alpha1 subunit mRNA and protein in DG at 1-2 weeks after SE. Enhanced alpha1 expression in DG resulted in a threefold increase in mean seizure-free time after SE and a 60% decrease in the number of rats developing epilepsy (recurrent spontaneous seizures) in the first 4 weeks after SE. These findings provide the first direct evidence that altering GABR subunit expression can affect the development of epilepsy and suggest that alpha1 subunit levels are important determinants of inhibitory function in hippocampus.


Subject(s)
Disease Models, Animal , Epilepsy, Temporal Lobe/metabolism , Epilepsy, Temporal Lobe/prevention & control , Parahippocampal Gyrus/metabolism , Protein Subunits/metabolism , Receptors, GABA-A/metabolism , Animals , Epilepsy, Temporal Lobe/genetics , Genetic Vectors/genetics , Humans , Male , Neural Inhibition/physiology , Protein Subunits/biosynthesis , Protein Subunits/genetics , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/biosynthesis , Receptors, GABA-A/genetics
8.
Epilepsia ; 47(10): 1665-73, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17054689

ABSTRACT

PURPOSE: Previous studies in neonatal (postnatal day 10) and adult rats suggest that status epilepticus (SE) induces changes in the alpha1 subunit of the GABA(A) receptor (GABRA1) in dentate granule neurons (DGNs) that are age dependent and vary inversely with the likelihood of epilepsy development. In the present study, we examined GABRA1 expression after SE at postnatal day 20 (P20), an intermediate age when only a subset of SE-exposed animals develop epilepsy. METHODS: SE was induced with lithium-pilocarpine or kainate at P20. Animals were video-EEG monitored after SE to determine the presence or absence of spontaneous seizures. GABRA1 mRNA and protein levels were determined 7 days or 3 months later in SE-exposed and control animals by using a combination of aRNA amplification, Western blotting, and immunohistochemistry techniques. RESULTS: GABRA1 mRNA levels in DGNs of SE-exposed rats that did not become epileptic were higher than those in control rats, but were not different from DGNs in epileptic SE-exposed rats. GABRA1 protein levels in dentate gyrus were significantly increased in both epileptic and nonepileptic SE-exposed rats compared with controls. GABRA1 mRNA changes were region specific and did not occur in CA1 or CA3 areas of hippocampus. GABRA1 alterations were present by 1 week after P20 SE and were similar whether pilocarpine or kainate was used to induced SE. CONCLUSIONS: P20 SE results in persistent increases in GABRA1 levels selectively in dentate gyrus. These changes preceded the onset of epilepsy, were not model specific, and occurred in both epileptic and nonepileptic animals.


Subject(s)
Dentate Gyrus/metabolism , Hippocampus/metabolism , Receptors, GABA-A/genetics , Status Epilepticus/genetics , Status Epilepticus/metabolism , Animals , Animals, Newborn , Dentate Gyrus/drug effects , Gene Expression , Hippocampus/drug effects , Kainic Acid/pharmacology , Lithium/pharmacology , Nucleic Acid Amplification Techniques , Pilocarpine/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/metabolism , Status Epilepticus/chemically induced
9.
J Biol Chem ; 281(40): 29431-5, 2006 Oct 06.
Article in English | MEDLINE | ID: mdl-16901909

ABSTRACT

Altered function of gamma-aminobutyric acid type A receptors (GABA(A)Rs) in dentate granule cells of the hippocampus has been associated with temporal lobe epilepsy (TLE) in humans and in animal models of TLE. Such altered receptor function (including increased inhibition by zinc and lack of modulation by benzodiazepines) is related, in part, to changes in the mRNA levels of certain GABA(A)R subunits, including alpha4, and may play a role in epileptogenesis. The majority of GABA(A)Rs that contain alpha4 subunits are extra-synaptic due to lack of the gamma2 subunit and presence of delta. However, it has been hypothesized that seizure activity may result in expression of synaptic receptors with altered properties driven by an increased pool of alpha4 subunits. Results of our previous work suggests that signaling via protein kinase C (PKC) and early growth response factor 3 (Egr3) is the plasticity trigger for aberrant alpha4 subunit gene (GABRA4) expression after status epilepticus. We now report that brain derived neurotrophic factor (BDNF) is the endogenous signal that induces Egr3 expression via a PKC/MAPK-dependent pathway. Taken together with the fact that blockade of tyrosine kinase (Trk) neurotrophin receptors reduces basal GABRA4 promoter activity by 50%, our findings support a role for BDNF as the mediator of Egr3-induced GABRA4 regulation in developing neurons and epilepsy and, moreover, suggest that BDNF may alter inhibitory processing in the brain by regulating the balance between phasic and tonic inhibition.


Subject(s)
Brain-Derived Neurotrophic Factor/physiology , Early Growth Response Protein 3/biosynthesis , Hippocampus/metabolism , Neurons/metabolism , Protein Subunits/metabolism , Receptors, GABA-A/metabolism , Animals , Cells, Cultured , Early Growth Response Protein 3/genetics , Hippocampus/cytology , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley
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