Phosphorylation of CRN2 by CK2 regulates F-actin and Arp2/3 interaction and inhibits cell migration

Xavier, Charles-Peter; Rastetter, Raphael H.; Blomacher, Margit; Stumpf, Maria; Himmel, Mirko; Morgan, Reginald O.; Fernández, Mónica; Wang, Conan K.; Osman, Asiah; Miyata, Yoshihiko; Gjerset, Ruth A.; Eichinger, Ludwig M.; Hofmann, Andreas; Linder, Stefan; Noegel, Angelika A.; Clemen, Christoph S.

doi:10.1038/srep00241

Cited by 37 publications

(43 citation statements)

References 83 publications

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“…Furthermore, we used cells with presence of the endogenous CRN2 (GFP) and over-expression of GFP-tagged CRN2 in addition to the endogenous CRN2 (GFP-CRN2). Moreover, we included cells that expressed phospho-resistant S463A (CRN2-shRNA/GFP-CRN2-S463A) and phospho-mimetic S463D mutant CRN2 (CRN2-shRNA/GFP-CRN2-S463D) in the knock-down background, based on the previous finding that phosphorylation at S463 inhibits the cellular activity of CRN2 12 (Table 1, columns 1-3). Expression of GFP-tagged wild-type or mutant CRN2 in U373 cells lacking endogenous CRN2 allows the analysis of phosphorylation-specific effects without interference of the endogenous protein.…”

Section: Discussionmentioning

confidence: 99%

“…Fourth, for U373 cells either expressing GFP alone or wild-type, phospho-resistant S463A, or phospho-mimetic S463D variants of GFP-CRN2 fusion proteins in the background of the knock-down of the endogenous CRN2, first, the puromycin selection cassette of pLKO.1-CMV-EGFP-MCS-puro was removed by BamHI/NsiI digestion and replaced by a neomycin cassette retrieved as PCR product from pEGFP-C1 with adjacent BamHI and NsiI restriction sites, resulting in pLKO.1-NEO-CMV-EGFP-MCS. Second, the CRN2-cassettes, which are resistant to the above shRNA oligo and code for either serine, aspartic acid, or alanine at position 463, were retrieved as PstI/SmaI fragments from pEGFP-CRN2res-WT, pEGFP-CRN2res-S463A, and pEGFP-CRN2res-S463D, 12 respectively, and transferred to the PstI/EcoRV opened pLKO.1-NEO-CMV-EGFP-MCS. Finally, after insertion of the primer duplex as in the third, constructs pLKO.1-NEO-CMV-EGFP-CRN2res-WT, pLKO.1-NEO-CMV-EGFP-CRN2res-S463A, and pLKO.1-NEO-CMV-EGFP-CRN2res-S463D were obtained.…”

Section: Methodsmentioning

confidence: 99%

“…9–12 CRN2's F-actin bundling activity and inhibitory effect on actin polymerization are abolished by CK2-dependent phosphorylation of CRN2 at serine 463, as shown by in vitro and in vivo kinase assays and the use of S463D phospho-mimetic and S463A phospho-resistant CRN2 variants. 12 In addition, CRN2 regulates the actin cytoskeleton via interaction with small G-proteins. An interaction of CRN2 with GDP-Rab27a increased its F-actin bundling activity and endocytosis of the insulin secretory membrane in pancreatic beta cells.…”

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CRN2 enhances the invasiveness of glioblastoma cells

et al. 2013

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BackgroundMovement of tumor cells involves dynamic remodeling of the actin cytoskeleton, which is regulated by actin binding proteins, such as CRN2 (synonyms: coronin 1C, coronin 3). In vitro, CRN2 participates in secretion, matrix degradation, protrusion formation, and cell migration. Furthermore, expression of CRN2 correlates with the malignant phenotype of human diffuse gliomas. CRN2's effects on actin polymerization and F-actin bundling are abolished by protein kinase 2 (CK2) dependent phosphorylation at serine 463.MethodsWe generated human U373 glioblastoma cell lines with knock-down of CRN2 or over-expression of CRN2 variants and studied their behavior in vitro and ex vivo in organotypic brain slice cultures.ResultsCRN2 over-expression and expression of the S463A phospho-resistant CRN2 variant increase proliferation, matrix degradation, and invasion but decrease adhesion and formation of invadopodia-like extensions in vitro. Knock-down of CRN2 and expression of S463D phospho-mimetic CRN2 generally have opposite effects. Analysis of invadopodia-like cell extensions shows a diffuse relocalization of F-actin in CRN2 knockdown cells, whereas expression of S463A and S463D mutant CRN2 causes enrichments of F-actin structures at the center and rime zone, respectively. Fluorescence recovery after photobleaching studies of CRN2 and F-actin in lamellipodia show that both CRN2 variants decrease the turnover of actin filaments. Glioblastoma cells over-expressing wild-type or S463A CRN2, which were transplanted onto brain slices, characteristically developed into tumors with an invasive phenotype.ConclusionsOverall, our data indicate that CRN2 participates in cancer progression via modulation of the actin cytoskeleton.

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confidence: 99%

Section: Methodsmentioning

confidence: 99%

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CRN2 enhances the invasiveness of glioblastoma cells

et al. 2013

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“…CK2 is also membrane targeted and acts directly on actin or actin-associated proteins such as WASP (Cory et al. , 2003) and CRN2 (Xavier et al. , 2012), regulating actin polymerization.…”

Section: Introductionmentioning

confidence: 99%

Protein kinase CK2 phosphorylates and activates p21-activated kinase 1

Shin

Kim

2013

MBoC

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“…17 Moreover, sequence scanning of ATG16L1 indicated that several serine and threonine residues are highly conserved and surrounded by acidic amino acids ( Fig. 2A), which is a signature motif (S/TXXE) for CSNK2, 18 we Figure 1. H/R increased autophagy and promoted ATG16L1 phosphorylation in cultured cardiomyocytes.…”

Section: Csnk2 Was Responsible For Atg16l1 Phosphorylation In H/rtreamentioning

confidence: 99%

ATG16L1 phosphorylation is oppositely regulated by CSNK2/casein kinase 2 and PPP1/protein phosphatase 1 which determines the fate of cardiomyocytes during hypoxia/reoxygenation

Song¹,

Wang³

et al. 2015

Autophagy

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(2015) ATG16L1 phosphorylation is oppositely regulated by CSNK2/casein kinase 2 and PPP1/protein phosphatase 1 which determines the fate of cardiomyocytes during hypoxia/reoxygenation, Autophagy, 11:8, 1308-1325, DOI: 10.1080/15548627.2015 Keywords: ATG16L1, autophagy, cardiomyocyte, casein kinase 2, protein phosphatase 1 Abbreviations: ACTB, actinb; AMPK, AMP activated protein kinase; ATG, autophagy-related; BCL2, B-cell CLL/lymphoma 2; BECN1/Beclin 1, autophagy related; CD, Crohn disease; CSNK2, casein kinase 2; GFP, green fluorescent protein; GNB2L1/ RACK1, guanine nucleotide binding protein (G protein)bpolypeptide 2-like 1; GST, glutathione S-transferase; H/R, hypoxia/reoxygenation; I/R, ischemia/reperfusion; LAD, left anterior descending; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 b; mRFP, monomeric red fluorescent protein; MTORC1, mechanistic target of rapamycin complex 1; NRVCs, neonatal rat ventricular cardiomyocytes; OA, okadaic acid; PE, phosphatidylethanolamine; PPP1, protein phosphatase 1; PPP2, protein phosphatase 2; PVDF, polyvinylidenedifluoride; SNP, single nucleotide polymorphism; SUPT20H/p38IP, suppressor of Ty 20 homolog (S. cerevisiae); TBB, 4,5,6,7-tetrabromobenzotriazole; lPP, l protein phosphatase.Recent studies have shown that the phosphorylation and dephosphorylation of ULK1 and ATG13 are related to autophagy activity. Although ATG16L1 is absolutely required for autophagy induction by affecting the formation of autophagosomes, the post-translational modification of ATG16L1 remains elusive. Here, we explored the regulatory mechanism and role of ATG16L1 phosphorylation for autophagy induction in cardiomyocytes. We showed that ATG16L1 was a phosphoprotein, because phosphorylation of ATG16L1 was detected in rat cardiomyocytes during hypoxia/ reoxygenation (H/R). We not only demonstrated that CSNK2 (casein kinase 2) phosphorylated ATG16L1, but also identified the highly conserved Ser139 as the critical phosphorylation residue for CSNK2. We further established that ATG16L1 associated with the ATG12-ATG5 complex in a Ser139 phosphorylation-dependent manner. In agreement with this finding, CSNK2 inhibitor disrupted the ATG12-ATG5-ATG16L1 complex. Importantly, phosphorylation of ATG16L1 on Ser139 was responsible for H/R-induced autophagy in cardiomyocytes, which protects cardiomyocytes from apoptosis. Conversely, we determined that wild-type PPP1 (protein phosphatase 1), but not the inactive mutant, associated with ATG16L1 and antagonized CSNK2-mediated phosphorylation of ATG16L1. Interestingly, one RVxF consensus site for PPP1 binding in the C-terminal tail of ATG16L1 was identified; mutation of this site disrupted its association with ATG16L1. Notably, CSNK2 also associated with PPP1, but ATG16L1 depletion impaired the interaction between CSNK2 and PPP1. Collectively, these data identify ATG16L1 as a bona fide physiological CSNK2 and PPP1 substrate, which reveals a novel molecular link from CSNK2 to activation of the autophagy-specific ATG12-ATG5-ATG16L1 complex and autoph...

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Phosphorylation of CRN2 by CK2 regulates F-actin and Arp2/3 interaction and inhibits cell migration

Cited by 37 publications

References 83 publications

CRN2 enhances the invasiveness of glioblastoma cells

CRN2 enhances the invasiveness of glioblastoma cells

Protein kinase CK2 phosphorylates and activates p21-activated kinase 1

ATG16L1 phosphorylation is oppositely regulated by CSNK2/casein kinase 2 and PPP1/protein phosphatase 1 which determines the fate of cardiomyocytes during hypoxia/reoxygenation

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