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1.  Domain Characterization of Rabbit Skeletal Muscle Myosin Light Chain Kinase 
The Journal of biological chemistry  1990;265(3):1724-1730.
Myosin light chain kinase can be divided into three distinct structural domains, an amino-terminal “tail,” of unknown function, a central catalytic core and a carboxy-terminal calmodulin-binding regulatory region. We have used a combination of deletion mutagenesis and monoclonal antibody epitope mapping to define these domains more closely. A 2.95-kilobase cDNA has been isolated that includes the entire coding sequence of rabbit skeletal muscle myosin light chain kinase (607 amino acids). This cDNA, expressed in COS cells encoded a Ca2+/calmodulin-dependent myosin light chain kinase with a specific activity similar to that of the enzyme purified from rabbit skeletal muscle. Serial carboxy-terminal deletions of the regulatory and catalytic domains were constructed and expressed in COS cells. The truncated kinases had no detectable myosin light chain kinase activity. Monoclonal antibodies which inhibit the activity of the enzyme competitively with respect to myosin light chain were found to bind between residues 235–319 and 165–173, amino-terminal of the previously defined catalytic core. Thus, residues that are either involved in substrate binding or in close proximity to a light chain binding site may be located more amino-terminal than the previously defined catalytic core.
PMCID: PMC4325999  PMID: 1688558
2.  Unfolding-resistant Translocase Targeting 
The Journal of biological chemistry  2005;280(29):27375-27382.
Heterotrimeric serine/threonine protein phosphatase 2A (PP2A) consists of scaffolding (A), catalytic (C), and variable (B, B’, and B”) subunits. Variable subunits dictate subcellular localization and substrate specificity of the PP2A holoenzyme. The Bβ regulatory subunit gene is mutated in spinocerebellar ataxia type 12, and one of its splice variants, Bβ2, targets PP2A to mitochondria to promote apoptosis in PC12 cells. Here, we report that Bβ2 is localized to the outer mitochondrial membrane by a novel mechanism, combining a cryptic mitochondrial import signal with a structural arrest domain. Scanning mutagenesis demonstrates that basic and hydrophobic residues mediate mitochondrial association and the proapoptotic activity of Bβ2. When fused to green fluorescent protein, the N terminus of Bβ2 acts as a cleavable mitochondrial import signal. Surprisingly, full-length Bβ2 is not detectably cleaved and is retained at the outer mitochondrial membrane, even though it interacts with the TOM22 import receptor, as shown by luciferase complementation in intact cells. Mutations that open the C-terminal β-propeller of Bβ2 facilitate mitochondrial import, indicating that this rigid fold acts as a stop-transfer domain by resisting the partial unfolding step prerequisite for matrix translocation. Because hybrids of prototypical import and β-propeller domains recapitulate this behavior, we predict the existence of other similarly localized proteins and a selection against highly stable protein folds in the mitochondrial matrix. This unfolding-resistant targeting to the mitochondrial translocase is necessary but not sufficient for the proapoptotic activity of Bβ2, which also requires association with the rest of the PP2A holoenzyme.
doi:10.1074/jbc.M503693200
PMCID: PMC4323179  PMID: 15923182
3.  Role of the POZ Zinc Finger Transcription Factor FBI-1 in Human and Murine Adipogenesis 
The Journal of biological chemistry  2003;279(12):11711-11718.
Poxvirus zinc finger (POZ) zinc finger domain transcription factors have been shown to play a role in the control of growth arrest and differentiation in several types of mesenchymal cells but not, as yet, adipocytes. We found that a POZ domain protein, factor that binds to inducer of short transcripts-1 (FBI-1), was induced during both murine and human preadipocyte differentiation with maximal expression levels seen at days 2–4. FBI-1 mRNA was expressed in human adipose tissue with the highest levels found in samples from morbidly obese subjects. Murine cell lines constitutively expressing FBI-1 showed evidence for accelerated adipogenesis with earlier induction of markers of differentiation and enhanced lipid accumulation, suggesting that FBI-1 may be an active participant in the differentiation process. Consistent with the properties of this family of proteins in other cell systems, 3T3L1 cells stably overexpressing FBI-1 showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, FBI-1 reduced the transcriptional activity of the cyclin A promoter. Thus, FBI-1, a POZ zinc finger transcription factor, is induced during the early phases of human and murine preadipocyte differentiation where it may contribute to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation.
doi:10.1074/jbc.M310240200
PMCID: PMC4303998  PMID: 14701838
4.  The Peroxisome Proliferator-activated Receptor-γ Regulates Murine Pyruvate Carboxylase Gene Expression in Vivo and in Vitro* 
The Journal of biological chemistry  2005;280(29):27466-27476.
Pyruvate carboxylase (PC) plays a crucial role in various metabolic pathways, including gluconeogenesis, lipogenesis, and glucose-induced insulin secretion. Here we showed for the first time that the PC gene is transcriptionally regulated by peroxisome proliferator-activated receptor-γ (PPARγ) in vitro and in vivo in white and brown adipose tissue. PC mRNA and protein are markedly increased during differentiation of 3T3-L1 cells and HIB-1B, in parallel with the expression of the adipogenic transcription factors, CCAAT-enhancer binding protein α, PPARγ1, and PPARγ2. Tumor necrosis factor-α, a cytokine that blocks differentiation of 3T3-L1 cells, suppressed PC expression. Co-transfection studies in 3T3-L1 preadipocytes or HEK293T cells with a 2.3-kb promoter fragment of mouse PC gene linked to a luciferase reporter construct and with plasmids overexpressing retinoid X receptor α/PPARγ1 or retinoid X receptor α/PPARγ2 showed a 6–8-fold increase above the basal promoter activity. Furthermore, treatment of these transfected cells with the PPARγ agonist doubled the promoter activity. Mutation of the putative PPAR-response element-(−386/−374) of this 2.3-kb PC promoter fragment abolished the PPARγ response. Gel shift and chromatin immunoprecipitation assays demonstrated that endogenous PPARγ binds to this functional PPAR-response element of the PC promoter. Mice with targeted disruption of the PPARγ2 gene displayed ~50–60% reduction of PC mRNA and protein in white adipose tissue. Similarly, in brown adipose tissue of PPARγ2-deficient mice subjected to cold exposure, PC mRNA was 40% lower than that of wild type mice. Impaired in vitro differentiation of white adipocytes of PPARγ2 knock-out mice was also associated with a marked reduction of PC mRNA. Our findings identified PC as a PPARγ-regulated gene and suggested a role for PPARγ regulating intermediary metabolism.
doi:10.1074/jbc.M503836200
PMCID: PMC4304003  PMID: 15917242
5.  Identification and Cloning of Centaurin-α 
The Journal of biological chemistry  1996;271(31):18859-18868.
Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP4), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-α. Binding specificity was determined using displacement of 1-O-[3H](3-[4-benzoyldihydrocinnamidyl]propyl)-InsP4 photoaffinity labeling. Centaurin-α displayed highest affinity for phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) (IC50 = 120 nm), whereas InsP4, PtdInsP2, and InsP3 bound with 5-, 12-, and >50-fold lower affinity, respectively. Screening a rat brain cDNA library with a polymerase chain reaction product, generated using partial amino acid sequence from tryptic peptides, yielded a full-length clone. The 2,450-base pair cDNA contained an open reading frame (ORF) encoding a novel protein of 419 amino acids. Northern analysis revealed a 2.5-kilobase transcript that is highly expressed in brain. The deduced sequence contains a novel putative zinc finger motif, 10 ankyrin-like repeats, and shows homology to recently identified yeast and mammalian Arf GTPase-activating proteins. Given the specificity of binding and enrichment in brain, centaurin-α is a candidate PtdInsP3 receptor that may link the activation of phosphoinositide 3-kinase to downstream responses in the brain.
PMCID: PMC4298166  PMID: 8702546
6.  Regulation of Protein Phosphatase Inhibitor-1 by Cyclin-dependent Kinase 5* 
The Journal of biological chemistry  2007;282(22):16511-16520.
Inhibitor-1, the first identified endogenous inhibitor of protein phosphatase 1 (PP-1), was previously reported to be a substrate for cyclin-dependent kinase 5 (Cdk5) at Ser67. Further investigation has revealed the presence of an additional Cdk5 site identified by mass spectrometry and confirmed by site-directed mutagenesis as Ser6. Basal levels of phospho-Ser6 inhibitor-1, as detected by a phosphorylation state-specific antibody against the site, existed in specific regions of the brain and varied with age. In the striatum, basal in vivo phosphorylation and dephosphorylation of Ser6 were mediated by Cdk5, PP-2A, and PP-1, respectively. Additionally, calcineurin contributed to dephosphorylation under conditions of high Ca2+. In biochemical assays the function of Cdk5-dependent phosphorylation of inhibitor-1 at Ser6 and Ser67 was demonstrated to be an intramolecular impairment of the ability of inhibitor-1 to be dephosphorylated at Thr35; this effect was recapitulated in two systems in vivo. Dephosphorylation of inhibitor-1 at Thr35 is equivalent to inactivation of the protein, as inhibitor-1 only serves as an inhibitor of PP-1 when phosphorylated by cAMP-dependent kinase (PKA) at Thr35. Thus, inhibitor-1 serves as a critical junction between kinase- and phosphatase-signaling pathways, linking PP-1 to not only PKA and calcineurin but also Cdk5.
doi:10.1074/jbc.M701046200
PMCID: PMC4296900  PMID: 17400554
7.  [No title available] 
PMCID: PMC4295213  PMID: 8382698
8.  [No title available] 
PMCID: PMC4293020  PMID: 16601116
9.  [No title available] 
PMCID: PMC4291123  PMID: 14532282
10.  [No title available] 
PMCID: PMC4291127  PMID: 12235138
11.  Protein Kinase A-mediated Phosphorylation Modulates Cytochrome c Oxidase Function and Augments Hypoxia and Myocardial Ischemia-related Injury* 
The Journal of biological chemistry  2005;281(4):2061-2070.
We have investigated the effects of hypoxia and myocardial ischemia/reperfusion on the structure and function of cytochrome c oxidase (CcO). Hypoxia (0.1% O2 for 10 h) and cAMP-mediated inhibition of CcO activity were accompanied by hyperphosphorylation of subunits I, IVi1, and Vb and markedly increased reactive O2 species production by the enzyme complex in an in vitro system that uses reduced cytochrome c as an electron donor. Both subunit phosphorylation and enzyme activity were effectively reversed by 50 nm H89 or 50 nm myristoylated peptide inhibitor (MPI), specific inhibitors of protein kinase A, but not by inhibitors of protein kinase C. In rabbit hearts subjected to global and focal ischemia, CcO activity was inhibited in a time-dependent manner and was accompanied by hyperphosphorylation as in hypoxia. Additionally, CcO activity and subunit phosphorylation in the ischemic heart were nearly completely reversed by H89 or MPI added to the perfusion medium. Hyperphosphorylation of subunits I, IVi1, and Vb was accompanied by reduced subunit contents of the immunoprecipitated CcO complex. Most interestingly, both H89 and MPI added to the perfusion medium dramatically reduced the ischemia/reperfusion injury to the myocardial tissue. Our results pointed to an exciting possibility of using CcO activity modulators for controlling myocardial injury associated with ischemia and oxidative stress conditions.
doi:10.1074/jbc.M507741200
PMCID: PMC4271455  PMID: 16303765
12.  FGF Homologous Factors and the Islet Brain-2 Scaffold Protein Regulate Activation of a Stress-Activated Protein Kinase 
The Journal of biological chemistry  2002;277(51):49111-49119.
Fibroblast growth factor homologous factors (FHFs) form native intracellular complexes with the mitogen-activated protein kinase (MAPK) scaffold protein islet-brain 2 (IB2) in adult brain. FHF binding to IB2 facilitates recruitment of the MAPK p38δ (SAPK4), while failing to stimulate binding of JNK, the preferred kinase of the related scaffold IB1 (JIP-1). We now report further biochemical evidence supporting FHFs as regulators of IB2’s function as a scaffold for p38δ kinase activation. MLK3 and IB2 synergistically activate p38δ, but not the MAPKs JNK-1 and p38α. Binding of p38δ to IB2 is mediated by the carboxyterminal half of the scaffold (IB2Δ1–436). FHF2 also binds weakly to IB2Δ1–436, and can thereby increase p38δ interaction with IB2Δ1–436. FHF-induced recruitment of p38δ to IB2 is accompanied by increased levels of activated p38δ, and synergistic activation of p38δ by MLK3/IB2 is further enhanced by FHF2. Consistent with a role for FHFs as signaling molecules, FHF2 isolated from rat brain is serine/threonine phosphorylated, and FHF can serve as a substrate for p38δ in vitro. These results support the existence of a signaling module in which IB2 scaffolds a MLK3/MKK/p38δ kinase cascade. FHFs aid in recruitment of p38 to IB2 and may serve as kinase substrates.
doi:10.1074/jbc.M205520200
PMCID: PMC4266389  PMID: 12244047
13.  Cyclin D3 Expression in Melanoma Cells Is Regulated by Adhesion-dependent Phosphatidylinositol 3-Kinase Signaling and Contributes to G1-S Progression* # 
The Journal of biological chemistry  2006;281(35):25644-25651.
D-type cyclins regulate G1 cell cycle progression by enhancing the activities of cyclin-dependent kinases (CDKs), and their expression is frequently altered in malignant cells. We and others have previously shown that cyclin D1 is up-regulated in melanoma cells through adhesion-independent MEK-ERK1/2 signaling initiated by mutant B-RAF. Here, we describe the regulation and role of cyclin D3 in human melanoma cells. Cyclin D3 expression was enhanced in a cell panel of human melanoma cell lines compared with melanocytes and was regulated by fibronectin-mediated phosphatidylinositol 3-kinase/Akt signaling but not MEK activity. RNA interference experiments demonstrated that cyclin D3 contributed to G1-S cell cycle progression and proliferation in melanoma cells. Overexpression of cyclin D1 did not recover the effects of cyclin D3 knockdown. Finally, immunoprecipitation studies showed that CDK6 is a major binding partner for cyclin D3, whereas CDK4 preferentially associated with cyclin D1. Together, these findings demonstrate that cyclin D3 is an important regulator of melanoma G1-S cell cycle progression and that D-type cyclins are differentially regulated in melanoma cells.
doi:10.1074/jbc.M600197200
PMCID: PMC4266577  PMID: 16815849
14.  MOLECULAR ARCHITECTURE AND DIVALENT CATION ACTIVATION OF TvoK, A PROKARYOTIC POTASSIUM CHANNEL* 
The Journal of biological chemistry  2007;282(33):24302-24309.
RCK domains form a family of ligand-binding domains found in many prokaryotic K+ channels and transport proteins. While many RCK domains contain an apparent nucleotide binding motif, some are known instead to bind Ca2+, which can then facilitate channel opening. Here we report on the molecular architecture and ligand-activation properties of an RCK-containing potassium channel cloned from the prokaryote Thermoplasma volcanium. This channel, called TvoK, is of an apparent molecular mass and subunit composition that is consistent with the hetero-octameric configuration hypothesized for the related MthK channel, in which four channel-tethered RCK domains coassemble with four soluble (untethered) RCK domains. The expression of soluble TvoK RCK subunits arises from an unconventional UUG start codon within the TvoK gene; silent mutagenesis of this alternative start codon abolishes expression of the soluble form of the TvoK RCK domain. Using single-channel recording of purified, reconstituted TvoK, we found that the channel is activated by Ca2+, as well as Mg2+, Mn2+ and Ni2+. This non-selective divalent activation is in contrast with the activation properties of MthK, which is selectively activated by Ca2+. Transplantation of the TvoK RCK domain into MthK generates a channel that can be activated by Mg2+, illustrating that the Mg2+ binding site is likely contained within the RCK domain. We present a working hypothesis for TvoK gating in which the binding of either Ca2+ or Mg2+ can contribute ~5 kcal/mole toward stabilization of the channel’s open conformation.
doi:10.1074/jbc.M703650200
PMCID: PMC4259576  PMID: 17588939
15.  V-ATPase V0 Sector Subunit a1 in Neurons Is a Target of Calmodulin*,S 
The Journal of biological chemistry  2007;283(1):294-300.
The V0 complex forms the proteolipid pore of a vesicular ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been suggested in vacuolar fusion in yeast and synaptic vesicle exocytosis in fly neurons. Evidence for a direct role in secretion has also recently been presented in mouse and worm. The molecular mechanisms of how the V0 components might act or are regulated are largely unknown. Here we report the identification and characterization of a calmodulin-binding site in the large cytosolic N-terminal region of the Drosophila protein V100, the neuron-specific V0 subunit a1. V100 forms a tight complex with calmodulin in a Ca2+-dependent manner. Mutations in the calmodulin-binding site in Drosophila lead to a loss of calmodulin recruitment to synapses. Neuronal expression of a calmodulin-binding deficient V100 uncovers an incomplete rescue at low levels and cellular toxicity at high levels. Our results suggest a vesicular ATPase V0-dependent function of calmodulin at synapses.
doi:10.1074/jbc.M708058200
PMCID: PMC4249936  PMID: 17933871
16.  Specificity and Autoregulation of Notch Binding by Tandem WW Domains in Suppressor of Deltex 
The Journal of biological chemistry  2007;282(39):29032-29042.
WW domains target proline-tyrosine (PY) motifs and frequently function as tandem pairs. When studied in isolation, single WW domains are notably promiscuous and regulatory mechanisms are undoubtedly required to ensure selective interactions. Here, we show that the fourth WW domain (WW4) of Suppressor of Deltex, a modular Nedd4-like protein that down-regulates the Notch receptor, is the primary mediator of a direct interaction with a Notch-PY motif. A natural Trp to Phe substitution in WW4 reduces its affinity for general PY sequences and enhances selective interaction with the Notch-PY motif via compensatory specificity-determining interactions with PY-flanking residues. When WW4 is paired with WW3, domain-domain association, impeding proper folding, competes with Notch-PY binding to WW4. This novel mode of autoinhibition is relieved by binding of another ligand to WW3. Such cooperativity may facilitate the transient regulatory interactions observed in vivo between Su(dx) and Notch in the endocytic pathway. The highly conserved tandem arrangement of WW domains in Nedd4 proteins, and similar arrangements in more diverse proteins, suggests domain-domain communication may be integral to regulation of their associated cellular activities.
doi:10.1074/jbc.M703453200
PMCID: PMC4244684  PMID: 17656366
17.  Dual Roles for Lipolysis and Oxidation in Peroxisome Proliferation-Activator Receptor Responses to Electronegative Low Density Lipoprotein* 
The Journal of biological chemistry  2003;278(41):39874-39881.
Low density lipoprotein (LDL) exists in various forms that possess unique characteristics, including particle content and metabolism. One circulating subfraction, electronegative LDL (LDL(−)), which is increased in familial hypercholesterolemia and diabetes, is implicated in accelerated atherosclerosis. Cellular responses to LDL(−) remain poorly described. Here we demonstrate that LDL(−) increases tumor necrosis factor α (TNFα)–induced inflammatory responses through NFκB and AP-1 activation with corresponding increases in vascular cell adhesion molecule-1 (VCAM1) expression. LDL receptor overexpression increased these effects. In contrast, exposing LDL(−) to the key lipolytic enzyme lipoprotein lipase (LPL) reversed these responses, inhibiting VCAM1 below levels seen with TNFα alone. LPL is known to act on lipoproteins to generate endogenous peroxisomal proliferator-activated receptor α (PPARα) ligand, thus limiting inflammation. These responses varied according to the lipoprotein substrate triglyceride content (very low density lipoprotein ≫ LDL > high density lipoprotein). The PPARα activation seen with LDL, however, was disproportionately high. We show here that MUT LDL activates PPARα to an extent proportional to its LDL(−) content. As compared with LDL(−) alone, LPL-treated LDL(−) increased PPARα activation 20-fold in either cell-based transfection or radioligand displacement assays. LPL-treated LDL(−) suppressed NFκB and AP-1 activation, increasing expression of the PPARα target gene IκBα, although only in the genetic presence of PPARα and with intact LPL hydrolysis. Mass spectrometry reveals that LPL-treatment of either LDL or LDL(−) releases hydroxy-octadecadienoic acids (HODEs), potent PPARα activators. These findings suggest LPL-mediated PPARα activation as an alternative catabolic pathway that may limit inflammatory responses to LDL(−).
doi:10.1074/jbc.M306786200
PMCID: PMC4231711  PMID: 12878589
18.  Flow Shear Stress Stimulates Gab1 Tyrosine Phosphorylation to Mediate Protein Kinase B and Endothelial Nitric-oxide Synthase Activation in Endothelial Cells* 
The Journal of biological chemistry  2005;280(13):12305-12309.
Fluid shear stress generated by blood flow modulates endothelial cell function via specific intracellular signaling events. We showed previously that flow activated the phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) via Src kinase-dependent transactivation of vascular endothelial growth factor receptor 2 (VEGFR2). The scaffold protein Gab1 plays an important role in receptor tyrosine kinase-mediated signal transduction. We found here that laminar flow (shear stress = 12 dynes/cm2) rapidly stimulated Gab1 tyrosine phosphorylation in both bovine aortic endothelial cells and human umbilical vein endothelial cells, which correlated with activation of Akt and eNOS. Gab1 phosphorylation as well as activation of Akt and eNOS by flow was inhibited by the Src kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and VEGFR2 kinase inhibitors SU1498 and VTI, suggesting that flow-mediated Gab1 phosphorylation is Src kinase-dependent and VEGFR2-dependent. Tyrosine phosphorylation of Gab1 by flow was functionally important, because flow stimulated the association of Gab1 with the PI3K subunit p85 in a time-dependent manner. Furthermore, transfection of a Gab1 mutant lacking p85 binding sites inhibited flow-induced activation of Akt and eNOS. Finally, knockdown of endogenous Gab1 by small interference RNA abrogated flow activation of Akt and eNOS. These data demonstrate a critical role of Gab1 in flow-stimulated PI3K/Akt/eNOS signal pathway in endothelial cells.
doi:10.1074/jbc.M500294200
PMCID: PMC4228934  PMID: 15665327
19.  A Novel Role of Vascular Endothelial Cadherin in Modulating c-Src Activation and Downstream Signaling of Vascular Endothelial Growth Factor* 
The Journal of biological chemistry  2008;283(11):7261-7270.
Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis and vascular permeability, in which c-Src tyrosine kinase plays an essential role. However, the mechanisms by which VEGF stimulates c-Src activation have remained unclear. Here, we demonstrate that vascular endothelial cadherin (VE-cadherin) plays a critical role in regulating c-Src activation in response to VEGF. In vascular endothelial cells, VE-cadherin was basally associated with c-Src and Csk (C-terminal Src kinase), a negative regulator of Src activation. VEGF stimulated Csk release from VE-cadherin by recruiting the protein tyrosine phosphatase SHP2 to VE-cadherin signaling complex, leading to an increase in c-Src activation. Silencing VE-cadherin with small interference RNA significantly reduced VEGF-stimulated c-Src activation. Disrupting the association of VE-cadherin and Csk through the reconstitution of Csk binding-defective mutant of VE-cadherin also diminished Src activation. Moreover, inhibiting SHP2 by small interference RNA and adenovirus-mediated expression of a catalytically inactive mutant of SHP2 attenuated c-Src activation by blocking the disassociation of Csk from VE-cadherin. Furthermore, VE-cadherin and SHP2 differentially regulates VEGF downstream signaling. The inhibition of c-Src, VE-cadherin, and SHP2 diminished VEGF-mediated activation of Akt and endothelial nitric-oxide synthase. In contrast, inhibiting VE-cadherin and SHP2 enhanced ERK1/2 activation in response to VEGF. These findings reveal a novel role for VE-cadherin in modulating c-Src activation in VEGF signaling, thus providing new insights into the importance of VE-cadherin in VEGF signaling and vascular function.
doi:10.1074/jbc.M702881200
PMCID: PMC4228940  PMID: 18180305
20.  Protein Kinase C-dependent Protein Kinase D Activation Modulates ERK Signal Pathway and Endothelial Cell Proliferation by Vascular Endothelial Growth Factor* 
The Journal of biological chemistry  2005;280(39):33262-33269.
Vascular endothelial growth factor (VEGF) is essential for many angiogenic processes both in normal conditions and in pathological conditions. However, the signaling pathways involved in VEGF-induced angiogenesis are not well defined. Protein kinase D (PKD), a newly described serine/threonine protein kinase, has been implicated in many signal transduction pathways and in cell proliferation. We hypothesized that PKD would mediate VEGF signaling and function in endothelial cells. Here we found that VEGF rapidly and strongly stimulated PKD phosphorylation and activation in endothelial cells via VEGF receptor 2 (VEGFR2). The pharmacological inhibitors for phospholipase Cγ (PLCγ) and protein kinase C (PKC) significantly inhibited VEGF-induced PKD activation, suggesting the involvement of the PLCγ/PKC pathway. In particular, PKCα was critical for VEGF-induced PKD activation since both overexpression of adenovirus PKCα dominant negative mutant and reduction of PKCα expression by small interfering RNA markedly inhibited VEGF-induced PKD activation. Importantly, we found that small interfering RNA knockdown of PKD and PKCα expression significantly attenuated ERK activation and DNA synthesis in endothelial cells by VEGF. Taken together, our results demonstrated for the first time that VEGF activates PKD via the VEGFR2/PLCγ/ PKCα pathway and revealed a critical role of PKD in VEGF-induced ERK signaling and endothelial cell proliferation.
doi:10.1074/jbc.M503198200
PMCID: PMC4228942  PMID: 16006559
21.  Regulation of the Interleukin-7 Receptor α Promoter by the Ets Transcription Factors PU.1 and GA-binding Protein in Developing B Cells* 
The Journal of biological chemistry  2007;282(19):14194-14204.
Signaling through the IL-7 receptor (IL-7R) is required for development and maintenance of the immune system. The receptor for IL-7 is heterodimeric, consisting of a common γ chain (γc, encoded by Il2rg) and an α subunit (IL-7Rα, encoded by Il7r). The Il7r gene is expressed specifically in the immune system in a developmental stage-specific manner. It is not known how the Il7r gene is transcriptionally regulated during B cell development. The goal of this study is to elucidate the function of the Il7r promoter region in developing B cells. Using a combination of 5′ rapid amplification of cDNA ends analysis, transient transfection assays, and DNase I hypersensitivity mapping, we identified the location of the Il7r promoter. Using a combination of electrophoretic mobility shift analysis, chromatin immunoprecipitation experiments, and RNA interference experiments, we found that the Ets transcription factors PU.1 and GA-binding protein (GABP) activate the Il7r promoter by interacting with a highly conserved Ets binding site. In committed B lineage cells, GABP can promote Il7r transcription in the absence of PU.1. However, the results of retroviral gene transfer experiments suggest that PU.1 is uniquely required to initiate transcription of the Il7r locus at the earliest stages of progenitor B cell generation. In summary, these results suggest that Il7r transcription is regulated by both PU.1 and GABP in developing B cells.
doi:10.1074/jbc.M700377200
PMCID: PMC4222673  PMID: 17392277
22.  Ethanol Inhibits L1-mediated Neurite Outgrowth in Postnatal Rat Cerebellar Granule Cells 
The Journal of biological chemistry  1999;274(19):13264-13270.
The neuropathology of the effects of ethanol on the developing central nervous system are similar to those of patients with mutations in L1, a neural cell adhesion molecule. This observation suggests that inhibition of L1 plays a role in the pathogenesis of alcohol-related neurodevelopmental disorders. Here we examine the effects of ethanol on L1 homophilic binding and on L1-mediated neurite outgrowth. Ethanol had no effect on cell adhesion or aggregation in a myeloma cell line expressing full-length human L1. In contrast, the rate of L1-mediated neurite outgrowth of rat postnatal day 6 cerebellar granule cells grown on a substratum of Ng-CAM, the chick homologue of L1, was inhibited by 48.6% in the presence of ethanol with a half-maximal concentration of 4.7mm. The same effect was found with soluble L1-Fc, thus showing that the inhibitory effect is not dependent on cell adhesion. In contrast, neither laminin nor N-cadherin-mediated neurite outgrowth was inhibited by physiologic concentrations of ethanol. We conclude that one mechanism of ethanol’s toxicity to the developing central nervous system may be the inhibition of L1-mediated neurite outgrowth.
PMCID: PMC4197854  PMID: 10224086
23.  Lysosomal Localization of TRPML3 Depends on TRPML2 and the Mucolipidosis-associated Protein TRPML1*S 
The Journal of biological chemistry  2006;281(25):17517-17527.
Mucolipidosis type IV is an autosomal recessive lysosomal storage disorder characterized by severe neurodegeneration, achlorhydria, and visual impairments such as corneal opacity and strabismus. The disease arises due to mutations in a group 2 transient receptor potential (TRP)-related cation channel, TRPML1. Mammals encode two additional TRPML proteins named TRPML2 and TRPML3. Information regarding the propensity of these proteins to multimerize, their subcellular distribution and mechanisms that regulate their trafficking are limited. Here we demonstrate that TRPMLs interact to form homo- and heteromultimers. Moreover, the presence of either TRPML1 or TRPML2 specifically influences the spatial distribution of TRPML3. TRPML1 and TRPML2 homo-multimers are lysosomal proteins, whereas TRPML3 homomultimers are in the endoplasmic reticulum. However, TRPML3 localizes to lysosomes when coexpressed with either TRPML1 or TRPML2 and is comparably mislocalized when lysosomal targeting of TRPML1 and TRPML2 is disrupted. Conversely, TRPML3 does not cause retention of TRPML1 or TRPML2 in the endoplasmic reticulum. These data demonstrate that there is a hierarchy controlling the subcellular distributions of the TRPMLs such that TRPML1 and TRPML2 dictate the localization of TRPML3 and not vice versa.
doi:10.1074/jbc.M600807200
PMCID: PMC4196876  PMID: 16606612
24.  The Hop Cassette of the PAC1 Receptor Confers Coupling to Ca2+ Elevation Required for Pituitary Adenylate Cyclase-activating Polypeptide-evoked Neurosecretion* 
The Journal of biological chemistry  2007;282(11):8079-8091.
We have identified the single PAC1 receptor variant responsible for Ca2+ mobilization from intracellular stores and influx through voltage-gated Ca2+ channels in bovine chromaffin cells and the domain of this receptor variant that confers coupling to [Ca2+]i elevation. This receptor (bPAC1hop) contains a 28-amino acid “hop” insertion in the third intracellular loop, with a full-length 171-amino acid N terminus. Expression of the bPAC1hop receptor in NG108–15 cells, which lack endogenous PAC1 receptors, reconstituted high affinity PACAP binding and PACAP-dependent elevation of both cAMP and intracellular Ca2+ concentrations ([Ca2+]i). Removal of the hop domain and expression of this receptor (bPAC1null) in NG108–15 cells reconstituted high affinity PACAP binding and PACAP-dependent cAMP generation but without a corresponding [Ca2+] i elevation. PC12-G cells express sufficient levels of PAC1 receptors to provide PACAP-saturable coupling to adenylate cyclase and to drive PACAP-dependent differentiation but do not express PAC1 receptors at levels found in postmitotic neuronal and endocrine cells and do not support PACAP-mediated neurosecretion. Expression of bPAC1hop, but not bPAC1null, at levels comparable with those of bPAC1hop in bovine chromaffin cells resulted in acquisition by PC12-G cells of PACAP-dependent [Ca2+] i increase and extracellular Ca2+ influx. In addition, PC12-G cells expressing bPAC1hop acquired the ability to release [3H] norepinephrine in a Ca2+ influx-dependent manner in response to PACAP. Expression of PACAP receptors in neuroendocrine rather than nonneuroendocrine cells reveals key differences between PAC1hop and PAC1null coupling, indicating an important and previously unrecognized role of the hop cassette in PAC1-mediated Ca2+ signaling in neuroendocrine cells.
doi:10.1074/jbc.M609638200
PMCID: PMC4183215  PMID: 17213203
25.  Three-dimensional Structure of the Truncated Core of the Saccharomyces cerevisiae Pyruvate Dehydrogenase Complex Determined from Negative Stain and Cryoelectron Microscopy Images* 
The Journal of biological chemistry  1992;267(34):24769-24775.
Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of α-keto acids, forms the central core to which the other components are attached. We have imaged by negative stain and cryoelectron microscopy the truncated dihydrolipoamide acetyltransferase core (60 subunits; Mr = 2.7 × 106) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. Using icosahedral particle reconstruction techniques, we determined its structure to 25 Å resolution. Although the model derived from the negative stain reconstruction was approximately 20% smaller than the model derived from the frozen-hydrated data, when corrected for the effects of the electron microscope contrast transfer functions, the reconstructions showed excellent correspondence. The pentagonal dodecahedron-shaped macromolecule has a maximum diameter, as measured along the 3-fold axis, of ~226 Å (frozen-hydrated value), and 12 large openings (~63 Å in diameter) on the 5-fold axes that lead into a large solvent-accessible cavity (~76–140 Å diameter). The 20 vertices consist of cone-shaped trimers, each with a flattened base on the outside of the structure and an apex directed toward the center. The trimers are interconnected by 20 Å thick “bridges” on the 2-fold axes. These studies also show that the highest resolution features apparent in the frozen-hydrated reconstruction are revealed in a filtered reconstruction of the stained molecule.
PMCID: PMC4167662  PMID: 1280269

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