Isolation, Cloning, and Characterization of a New Mammalian Coronin Family Member, Coroninse, Which Is Regulated within the Protein Kinase C Signaling Pathway

Parente, John A.; Chen, Xunsheng; Zhou, Cheng-Jing; Petropoulos, Ann C.; Chew, Catherine S.

doi:10.1074/jbc.274.5.3017

Cited by 35 publications

(38 citation statements)

References 36 publications

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“…Thus, it is possible that vivo functions were similarly compromised. Also, the transfection efficiency with parietal cells is low and not all parietal cells in primary culture respond to acid secretory agonists with obvious changes in cytoskeletal morphology (Parente et al, 1999). Clearly, new experimental approaches will be required to confirm or discount a direct role for cAMPdependent phosphorylation in lasp-1 translocation and to define the specific cellular activities that are modulated during this translocation process.…”

Section: Discussionmentioning

confidence: 99%

“…Exponentially growing cells were transfected with the pcDNA3 vector containing HA-tagged wild-type lasp-1 cDNA and cDNA from phosphorylation site mutants using Effectene (Qiagen) as previously described (Parente et al, 1999). Forty-eight hours later, cells were incubated with forskolin (10 µM, 15 minutes) or an equal volume of DMSO vehicle.…”

Section: In Vitro and In Vivo Phosphorylation Site Analysesmentioning

confidence: 99%

“…The sequences of all clones were confirmed prior to use (Medical College of GA Core DNA Sequencing Facility; ABI Prism 377 automated DNA sequencer; ABI Prism Cycle Sequencing Dye Terminator Ready Reaction kits). For transfections, plasmid DNA was isolated and purified with Qiagen Maxiprep Endo-Free kits as previously described (Parente et al, 1999).…”

Section: Dna Constructs and Bacterial Protein Expressionmentioning

confidence: 99%

“…Madin Darby kidney (MDCK) cells, passage 20-25, were cultured using standard conditions. Transfection of various plasmids into gastric cells and MDCK cells was accomplished using Effectene (Qiagen) as previously described (Parente et al, 1999).…”

Section: Cellular Modelsmentioning

confidence: 99%

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Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo

Chew

Chen

Parente³

et al. 2002

Journal of Cell Science

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110

139

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Lasp-1 has been identified as a signaling molecule that is phosphorylated upon elevation of [cAMP]i in pancreas, intestine and gastric mucosa and is selectively expressed in cells within epithelial tissues. In the gastric parietal cell, cAMP-dependent phosphorylation induces the partial translocation of lasp-1 to the apically directed F-actin-rich canalicular membrane, which is the site of active HCl secretion. Lasp-1 is an unusual modular protein that contains an N-terminal LIM domain, a C-terminal SH3 domain and two internal nebulin repeats. Domain-based analyses have recently categorized this protein as an epithelial representative of the nebulin family, which also includes the actin binding, muscle-specific proteins,nebulin, nebulette and N-RAP.In this study, we show that lasp-1 binds to non-muscle filamentous (F)actin in vitro in a phosphorylation-dependent manner. In addition, we provide evidence that lasp-1 is concentrated within focal complexes as well as in the leading edges of lamellipodia and the tips of filopodia in non-transformed gastric fibroblasts. In actin pull-down assays, the apparent Kd of bacterially expressed his-tagged lasp-1 binding to F-actin was 2 μM with a saturation stoichiometry of ∼1:7. Phosphorylation of recombinant lasp-1 with recombinant PKA increased the Kd and decreased the Bmax for lasp-1 binding to F-actin. Microsequencing and site-directed mutagenesis localized the major in vivo and in vitro PKA-dependent phosphorylation sites in rabbit lasp-1 to S99 and S146. BLAST searches confirmed that both sites are conserved in human and chicken homologues. Transfection of lasp-1 cDNA encoding for alanine substitutions at S99 and S146, into parietal cells appeared to suppress the cAMP-dependent translocation of lasp-1 to the intracellular canalicular region. In gastric fibroblasts, exposure to the protein kinase C activator, PMA, was correlated with the translocation of lasp-1 into newly formed F-actin-rich lamellipodial extensions and nascent focal complexes. Since lasp-1 does not appear to be phosphorylated by PKC,these data suggest that other mechanisms in addition to cAMP-dependent phosphorylation can mediate the translocation of lasp-1 to regions of dynamic actin turnover. The localization of lasp-1 to these subcellular regions under a range of experimental conditions and the phosphorylation-dependent regulation of this protein in F-actin rich epithelial cells suggests an integral and possibly cell-specific role in modulating cytoskeletal/membrane-based cellular activities.

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Section: Discussionmentioning

confidence: 99%

Section: In Vitro and In Vivo Phosphorylation Site Analysesmentioning

confidence: 99%

Section: Dna Constructs and Bacterial Protein Expressionmentioning

confidence: 99%

Section: Cellular Modelsmentioning

confidence: 99%

See 2 more Smart Citations

Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo

Chew

Chen

Parente³

et al. 2002

Journal of Cell Science

Self Cite

110

139

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“…Oxyntic mucosal cell fractions enriched for the other major cell types were obtained from the same density gradients by collecting fractions sedimenting at different densities (6). In some experiments, parietal cell fractions were placed in primary culture for up to 3 days as previously described (9,37). Parietal cell acid-secretory responses in isolated gastric glands were assessed by measuring the accumulation of the weak base AP (10).…”

Section: Methodsmentioning

confidence: 99%

IQGAPs are differentially expressed and regulated in polarized gastric epithelial cells

Chew

Okamoto²,

Chen³

et al. 2005

American Journal of Physiology-Gastrointestinal and Liver Physi

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. IQGAPs are differentially expressed and regulated in polarized gastric epithelial cells. Am J Physiol Gastrointest Liver Physiol 288: G376 -G387, 2005. First published September 30, 2004; doi:10.1152/ajpgi.00290.2004.-IQGAPs, GTPase-activating proteins with an IQ motif, are thought to regulate many actin cytoskeleton-based activities through interactions with Cdc42 and Rac. Recently, Cdc42 was implicated in regulation of gastric parietal cell HCl secretion, and IQGAP2 was immunolocalized with Cdc42 to F-actin-rich intracellular canalicular membranes of isolated gastric parietal cells in primary culture. Here we sought to define distribution and localization of IQGAP1 and IQGAP2 in major oxyntic (acid-secreting) gastric mucosal cell types and to determine whether secretory agonists modulate these proteins. Differential staining protocols were used to identify different cell populations (parietal, chief, surface/pit, and mucous neck cells) in semi-intact glands isolated from rabbit gastric mucosae and to characterize these same cells after dispersion and fractionation on isopycnic density gradients with simultaneous staining for F-actin, H ϩ -K ϩ -ATPase, and GSII lectinbinding sites. There was a pronounced increase in intracellular F-actin staining in dispersed chief cells, apparently from internalization of F-actin-rich apical membranes that normally abut the gland lumen. Therefore, other membrane-associated proteins might also be redistributed by disruption of cell-cell contacts. Western blot analyses were used to quantitate relative concentrations of IQGAPs in defined mucosal cell fractions, and gastric glands were used for in situ localizations. We detected uniform levels of IQGAP2 expression in oxyntic mucosal cells with predominant targeting to regions of cellcell contact and nuclei of all cell types. IQGAP2 was not detected in parietal cell intracellular canaliculi. IQGAP1 expression was variable and targeted predominantly to the cortex of chief and mucous neck cells. Parietal cells expressed little or no IQGAP1 vs. other mucosal cell types. Phosphoprotein affinity chromatography, isoelectric focusing, and phosphorylation site analyses indicated that both IQGAP1 and IQGAP2 are phosphoproteins potentially regulated by [Ca 2ϩ ]i/ PKC and cAMP signaling pathways, respectively. Stimulation of glands with carbachol, which elevates [Ca 2ϩ ]i and activates PKC, induced apparent translocation of IQGAP1, but not IQGAP2, to apical poles of chief (zymogen) and mucous neck cells. This response was mimicked by PMA but not by ionomycin or by elevation of [cAMP] i with forskolin. Our observations support a novel, PKC-dependent role for IQGAP1 in regulated exocytosis and suggest that IQGAP2 may play a more general role in regulating cell-cell interactions and possibly migration within the gastric mucosa. parietal cell; chief cell; protein kinase C; carbachol; actin THE RHO GTPASES Cdc42 and Rac1 regulate actin cytoskeletal dynamics by interacting with a number of effectors, including the IQGAPs (5, 15, 43), which are s...

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Coronin proteins as multifunctional regulators of the cytoskeleton and membrane trafficking

2005

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Coronins constitute an evolutionarily conserved family of WD-repeat actin-binding proteins, which can be clearly classified into two distinct groups based on their structural features. All coronins possess a conserved basic N-terminal motif and three to ten WD repeats clustered in one or two core domains. Dictyostelium and mammalian coronins are important regulators of the actin cytoskeleton, while the fly Dpod1 and the yeast coronin proteins crosslink both actin and microtubules. Apart from that, several coronins have been shown to be involved in vesicular transport. C. elegans POD-1 and Drosophila coro regulate the actin cytoskeleton, but also govern vesicular trafficking as indicated by mutant phenotypes. In both organisms, defects in cytoskeleton and trafficking lead to severe developmental defects ranging from abnormal cell division to aberrant formation of morphogen gradients. Finally, mammalian coronin 7 appears not to execute any cytoskeleton-related functions, but rather participates in regulating Golgi trafficking. Here, we review recent data providing more insight into molecular mechanisms underlying the regulation of F-actin structures, cytoskeletal rearrangements and intracellular membrane transport by coronin proteins and the way that they might link cytoskeleton with trafficking in development and disease.

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Isolation, Cloning, and Characterization of a New Mammalian Coronin Family Member, Coroninse, Which Is Regulated within the Protein Kinase C Signaling Pathway

Cited by 35 publications

References 36 publications

Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo

Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo

IQGAPs are differentially expressed and regulated in polarized gastric epithelial cells

Coronin proteins as multifunctional regulators of the cytoskeleton and membrane trafficking

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