Dataset Information

A quantitative proteomic view of cofilin phosphorylation and its modulation using the LIM kinase inhibitor Pyr1

ABSTRACT: LIM kinases are located at a strategic crossroad, downstream of several signaling pathways and upstream of effectors such as microtubules and the actin cytoskeleton.. Cofilin is the only LIM kinases substrate that is well described to date, and its phosphorylation on serine 3 by LIM Kinases controls cofilin actin-severing activity. Consequently, LIM Kinases inhibition leads to actin cytoskeleton disorganization and blockade of cell motility, which makes this strategy attractive in anticancer strategies. We have used proteomic approaches to investigate quantitatively and in detail the phosphorylation status of cofilin in myeloid tumor cell lines of murine and human origin. Our results show that under standard conditions, only a relatively small fraction (10 to 30% depending on the cell line) of cofilin is phosphorylated (including serine 3 phosphorylation). In addition, after a pharmacological inhibition of LIM kinases, a residual cofilin phosphorylation is observed on serine 3. Interestingly, this 2D gel based proteomic study identified new phosphorylation sites on cofilin, such as threonine 63, tyrosine 82 and serine 108.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Hélène Diemer

LAB HEAD: Sarah Cianférani

PROVIDER: PXD010167 | Pride | 2018-12-19

REPOSITORIES: Pride

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A quantitative proteomic analysis of cofilin phosphorylation in myeloid cells and its modulation using the LIM kinase inhibitor Pyr1.

Prudent Renaud R Demoncheaux Nathalie N Diemer Hélène H Collin-Faure Véronique V Kapur Reuben R Paublant Fabrice F Lafanechère Laurence L Cianférani Sarah S Rabilloud Thierry T

PloS one 20181214 12

LIM kinases are located at a strategic crossroad, downstream of several signaling pathways and upstream of effectors such as microtubules and the actin cytoskeleton. Cofilin is the only LIM kinases substrate that is well described to date, and its phosphorylation on serine 3 by LIM kinases controls cofilin actin-severing activity. Consequently, LIM kinases inhibition leads to actin cytoskeleton disorganization and blockade of cell motility, which makes this strategy attractive in anticancer trea ...[more]

PMID: 30550596

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Project description:SMCs express plasminogen activator inhibitor-1 (PAI-1), which regulates SMC function and vascular remodeling. However, whether PAI-1 controls SMC cytoskeletal dynamics and stiffness is unknown, and the causal role of PAI-1 in arterial stiffening is undefined. SMCs from human coronary arteries and aortae of wild-type vs. PAI-1-deficient mice were cultured with or without PAI-039, a specific PAI-1 inhibitor, after which cell stiffness was measured by atomic force microscopy, filamentous actin structures were assessed by confocal microscopy, and the activities cofilin, LIM domain kinase 1 (LIMK), slingshot homolog 1 (SSH), and AMP-activated protein kinase (AMPK) were measured. RNA sequencing was performed to determine the effects of PAI-039 on SMC gene expression. Effects of PAI-039 on aortic stiffness were assessed by pulse wave velocity. PAI-039 significantly reduced intrinsic stiffness of human SMCs, which was accompanied by significant decreases in cytoplasmic actin filaments. Similar effects were observed in wild-type, but not in PAI-1-deficient SMCs. Mechanistically, PAI-039 significantly increased the activity of cofilin, an actin depolymerase, in SMCs expressing PAI-1, but not in PAI-1-deficient cells. PAI-039 had no significant effects on LIMK or SSH activity. RNA-sequencing analysis suggested that PAI-039 up-regulates AMPK signaling in SMCs, which was confirmed by western blotting. Inhibition of AMPK prevented activation of cofilin by PAI-039. In mice, PAI-039 significantly decreased aortic stiffness without significantly altering peri-aortic fibrosis. PAI-039 decreases intrinsic SMC stiffness by reducing cytoplasmic stress fiber content. These effects are mediated by AMPK-dependent activation of cofilin. PAI-039 also decreases aortic stiffness in vivo. These findings suggest that PAI-1 is an important regulator of the SMC cytoskeleton and that pharmacologic inhibition of PAI-1 has potential to treat cardiovascular diseases mediated by accelerated arterial stiffening.

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