PKA and actin play critical roles as downstream effectors in MRP4-mediated regulation of fibroblast migration

Sinha, Chandrima; Ren, Aiming; Arora, Kavisha; Moon, Chang Suk; Yarlagadda, Sunitha; Woodrooffe, Koryse; Lin, Shibin; Schuetz, John D.; Ziady, Assem G.; Naren, Anjaparavanda P.

doi:10.1016/j.cellsig.2015.03.022

Cited by 13 publications

(25 citation statements)

References 40 publications

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“…PKA activity can be monitored in real time using FRET-based sensor for PKA called pmAKAR3 7,17 . To check the specificity of pmAKAR3 for PKA activity, we treated HEK293 cells overexpressing pmAKAR3 or the point mutant pmAKAR3-TA, which contains a threonine-to-alanine mutation at substrate region for PKA and therefore irresponsive to PKA phosphorylation, with 25 μM of forskolin, a cAMP inducing agent 2 .…”

Section: Representative Resultsmentioning

confidence: 99%

“…IncuCyte ™ –based high content microscopy can capture and analyze cell behaviors like cell migration in a more convenient and accurate manner 18 . The high content microscopy data demonstrated that the effect of MRP4 on fibroblast migration is completely abolished by disruption of actin cytoskeleton or by inhibition of PKA 17 .…”

Section: Introductionmentioning

confidence: 94%

“…Using this pmAKAR3, we demonstrated that the leading edge of migrating Mrp4 −/− fibroblast exhibit more polarized PKA activity than Mrp4 +/+ fibroblasts, which in turn increase the cortical actin formation at the cell front 17 . Together, these events results in better cellular polarization and faster directional cell migration in the absence of MRP4.…”

Section: Introductionmentioning

confidence: 97%

“…IPA is a useful tool to analyze protein-protein interaction (both structural and functional) and explore their contributions in particular physiological and pathological events, based on literature and experimental evidences 15,16 . IPA indicated that F-actin is a major downstream target of MRP4 in the context of cell migration where cAMP and cGMP are the key signaling molecules 17 . This data were further confirmed by high content microscopy.…”

Section: Introductionmentioning

confidence: 99%

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Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Sinha

Arora

Naren

2016

JoVE

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Multidrug resistance protein 4 (MRP4) is a member of ATP binding cassette transporter (ABC) family and an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, it can regulate multiple cyclic nucleotide-dependent cellular processes including cell migration1,2. Previously, we demonstrated that in the absence of MRP4, fibroblasts cell contain higher level of intracellular cyclic nucleotides and can migrate faster. In order to understand the underlying mechanisms, we adopted a direct but multifaceted approach. First, we isolated potential interacting protein complex of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by subjecting it to mass-spectrometry. After identifying these unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. Using this approach, we elucidated the potential role of this protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Next, using high content microscopy, we performed cell migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of actin cytoskeleton or by inhibition of cAMP-dependent kinase A (PKA). In order to visualize signaling modulations in a migrating cell in real time, we utilized FRET based sensor for measuring PKA activity and concluded the presence of more polarized PKA activity compared to Mrp4+/+ fibroblasts, near the leading edge of migrating Mrp4−/− fibroblast which in turn increases the cortical actin formation and augments the process of migration. Together, this direct but multistep approach enables us to identify the proteins acting downstream to MRP4 and provide us an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

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Section: Representative Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

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Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Sinha

Arora

Naren

2016

JoVE

Self Cite

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“…A number of the biological processes identified by GO analysis have been previously reported to be regulated by cAMP. These include mRNA splicing (64)(65)(66), spindle organization (67,68), fibroblast migration (69,70), lamellipodium assembly (71,72), apoptosis (73)(74)(75)(76), ATM signaling (77,78), gene silencing via microRNA (79)(80)(81)(82), T-cell selection (83), and cytoskeletal reorganization (84). This study suggests which molecular substrates might participate in the regulation of these processes.…”

Section: Biological Processes Regulated By Different Pde-regulatedmentioning

confidence: 99%

Analyses of PDE-regulated phosphoproteomes reveal unique and specific cAMP-signaling modules in T cells

Beltejar

Lau

Golkowski

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Specific functions for different cyclic nucleotide phosphodiesterases (PDEs) have not yet been identified in most cell types. Conventional approaches to study PDE function typically rely on measurements of global cAMP, general increases in cAMP-dependent protein kinase (PKA), or the activity of exchange protein activated by cAMP (EPAC). Although newer approaches using subcellularly targeted FRET reporter sensors have helped define more compartmentalized regulation of cAMP, PKA, and EPAC, they have limited ability to link this regulation to downstream effector molecules and biological functions. To address this problem, we have begun to use an unbiased mass spectrometry-based approach coupled with treatment using PDE isozyme-selective inhibitors to characterize the phosphoproteomes of the functional pools of cAMP/PKA/EPAC that are regulated by specific cAMP-PDEs (the PDE-regulated phosphoproteomes). In Jurkat cells we find multiple, distinct PDE-regulated phosphoproteomes that can be defined by their responses to different PDE inhibitors. We also find that little phosphorylation occurs unless at least two different PDEs are concurrently inhibited in these cells. Moreover, bioinformatics analyses of these phosphoproteomes provide insight into the unique functional roles, mechanisms of action, and synergistic relationships among the different PDEs that coordinate cAMP-signaling cascades in these cells. The data strongly suggest that the phosphorylation of many different substrates contributes to cAMP-dependent regulation of these cells. The findings further suggest that the approach of using selective, inhibitor-dependent phosphoproteome analysis can provide a generalized methodology for understanding the roles of different PDEs in the regulation of cyclic nucleotide signaling.

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Efflux Transporters

Jungsuwadee¹,

Vore²

2018

Comprehensive Toxicology

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PKA and actin play critical roles as downstream effectors in MRP4-mediated regulation of fibroblast migration

Cited by 13 publications

References 40 publications

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Analyses of PDE-regulated phosphoproteomes reveal unique and specific cAMP-signaling modules in T cells

Efflux Transporters

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