CCM2–CCM3 interaction stabilizes their protein expression and permits endothelial network formation

Draheim, Kyle; Li, Xiaofeng; Zhang, Rong; Fisher, Oriana S.; Villari, Giulia; Boggon, Titus J.; Calderwood, David A.

doi:10.1083/jcb.201407129

Cited by 49 publications

(56 citation statements)

References 39 publications

(86 reference statements)

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“…Mutations and deletions were introduced by QuikChange (Agilent Technologies) mutagenesis or PCR amplification and subcloning into pEGFP (Takara Bio Inc.) or pLENTI-CMV-Hygro-GFP (Draheim et al, 2015) vectors. Cdc42 DNA was kindly provided by Brian Rosenberg (Yale University) and subcloned into pmCherry (in-house vector derived from pEGFP).…”

Section: Dna Constructsmentioning

confidence: 99%

PAK6 targets to cell-cell adhesions via its N-terminus in a Cdc42-dependent manner to drive epithelial colony escape

Morse

Sun

Olberding

et al. 2015

Journal of Cell Science

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The six serine/threonine kinases in the p21-activated kinase (PAK) family are important regulators of cell adhesion, motility and survival. PAK6, which is overexpressed in prostate cancer, was recently reported to localize to cell-cell adhesions and to drive epithelial cell colony escape. Here we report that PAK6 targeting to cell-cell adhesions occurs through its N-terminus, requiring both its Cdc42/ Rac interactive binding (CRIB) domain and an adjacent polybasic region for maximal targeting efficiency. We find PAK6 localization to cell-cell adhesions is Cdc42-dependent, as Cdc42 knockdown inhibits PAK6 targeting to cell-cell adhesions. We further find the ability of PAK6 to drive epithelial cell colony escape requires kinase activity and is disrupted by mutations that perturb PAK6 cell-cell adhesion targeting. Finally, we demonstrate that all type II PAKs (PAK4, PAK5 and PAK6) target to cell-cell adhesions, albeit to differing extents, but PAK1 (a type I PAK) does not. Notably, the ability of a PAK isoform to drive epithelial colony escape correlates with its targeting to cell-cell adhesions. We conclude that PAKs have a broader role in the regulation of cell-cell adhesions than previously appreciated.

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Section: Dna Constructsmentioning

confidence: 99%

PAK6 targets to cell-cell adhesions via its N-terminus in a Cdc42-dependent manner to drive epithelial colony escape

Morse

Sun

Olberding

et al. 2015

Journal of Cell Science

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“…Based on experimental and clinical data, CCM1-3 proteins are physically associated in order of CCM1-CCM2 and CCM2-CCM3, and the complex is required for correct protein localization at endothelial cell-cell junctions, vascular tubular and network stability as well as cell survival and proliferation [10, 21, 22]. In particular, CCM2-CCM3 interaction is indicated as maintaining stability of CCM3 and CCM2 proteins and protection from proteosomal degradation and normal endothelial cell network formation [10]. A recent proteomic study found that CCM3 in the STRIPAK complex established mutually exclusive interactions with PIP2A, germinal center kinases III (Stk24, Stk25, Mst4) and cortical actin binding protein 2 (CTTNBP2).…”

Section: Introductionmentioning

confidence: 99%

“…It may participate in regulating VEGFR2 receptor stability, PIP2A enzyme association with membrane phospholipids like PIP2 and PIP3, apoptosis and endothelial cell growth [9, 19, 21, 22, 27, 60]. CCM3 localization and molecular interaction in both complexes is considered critical for understanding CCM3 function, potential overlap between CCM2 and CCM3 pathology (due to CCM2 and CCM3 direct interaction) and differences from CCM1 and CCM2 pathways (RhoA, Cdc42 signaling and actin rearrangement) [10, 21, 66, 74]. How CCM3 regulate the TJ complex assembly and permeability, which target and signaling pathways are critical for development of CCM3 lesion is still awaits clarification.…”

Section: Introductionmentioning

confidence: 99%

PDCD10 (CCM3) regulates brain endothelial barrier integrity in cerebral cavernous malformation type 3: role of CCM3-ERK1/2-cortactin cross-talk

et al. 2015

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Impairment of brain endothelial barrier integrity is critical for cerebral cavernous malformation (CCM) lesion development. The current study investigates changes in tight junction (TJ) complex organization when PDCD10 (CCM3) is mutated/depleted in human brain endothelial cells. Analysis of lesions with CCM3 mutation and brain endothelial cells transfected with CCM3 siRNA (CCM3-knockdown) showed little or no increase in TJ transmembrane and scaffolding proteins mRNA expression, but proteins levels were generally decreased. CCM3- knockdown cells had a redistribution of claudin-5 and occludin from the membrane to the cytosol with no alterations in protein turnover but with diminished protein-protein interactions with ZO-1 and ZO-1 interaction with the actin cytoskeleton. The most profound effect of CCM3 mutation/depletion was on an actin-binding protein, cortactin. CCM3 depletion caused cortactin Ser-phosphorylation, dissociation from ZO-1 and actin, redistribution to the cytosol and degradation. This affected cortical actin ring organization, TJ complex stability and consequently barrier integrity, with constant hyperpermeability to inulin. A potential link between CCM3 depletion and altered cortactin was tonic activation of MAP kinase ERK1/2. ERK1/2 inhibition increased cortactin expression and incorporation into the TJ complex and improved barrier integrity. This study highlights the potential role of CCM3 in regulating TJ complex organization and brain endothelial barrier permeability.

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“…First described in focal adhesion kinase (FAK) (Arold et al, 2002;Hayashi et al, 2002), the compact four-helix bundle FAT domain was subsequently identified in the Cas-family of proteins (Arold et al, 2002;Garron et al, 2009;Mace et al, 2011), in the focal adhesion protein vinculin (Arold et al, 2002), in G protein-coupled receptor kinase-interacting protein ArfGAP 1 (GIT1) (Schmalzigaug et al, 2007) and more recently in cerebral cavernous malformation protein 3 (CCM3) (Li et al, 2010). The FAT domain mediates FAK interaction with paxillin (Hayashi et al, 2002;Hoellerer et al, 2003), FAK intra-molecular interactions (BramiCherrier et al, 2014), CCM3 interaction with CCM2 (Draheim et al, 2015), and interactions between the Cas-family proteins and members of the SH2-containing protein (NSP) family -NSP1, NSP2 (also known as AND-34 and BCAR3) and NSP3 (also known as SHEP1 and CHAT) (Garron et al, 2009;Mace et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

The focal adhesion targeting domain of p130Cas confers a mechanosensing function

Bradbury

Turner

Mitchell

et al. 2017

Journal of Cell Science

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Members of the Cas family of focal adhesion proteins contain a highly conserved C-terminal focal adhesion targeting (FAT) domain. To determine the role of the FAT domain in these proteins, we compared wild-type exogenous NEDD9 with a hybrid construct in which the NEDD9 FAT domain had been exchanged for the p130Cas (also known as BCAR1) FAT domain. Fluorescence recovery after photobleaching (FRAP) revealed significantly slowed exchange of the fusion protein at focal adhesions and significantly slower twodimensional migration. No differences were detected in cell stiffness as measured using atomic force microscopy (AFM) and in cell adhesion forces measured with a magnetic tweezer device. Thus, the slowed migration was not due to changes in cell stiffness or adhesion strength. Analysis of cell migration on surfaces of increasing rigidity revealed a striking reduction of cell motility in cells expressing the p130Cas FAT domain. The p130Cas FAT domain induced rigiditydependent phosphorylation of tyrosine residues within NEDD9. This in turn reduced post-translational cleavage of NEDD9, which we show inhibits NEDD9-induced migration. Collectively, our data therefore suggest that the p130Cas FAT domain uniquely confers a mechanosensing function.

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CCM2–CCM3 interaction stabilizes their protein expression and permits endothelial network formation

Abstract: CCM2–CCM3 interactions protect CCM2 and CCM3 from proteasomal degradation, and both CCM2 and CCM3 are required for normal endothelial cell network formation.

Cited by 49 publications

References 39 publications

PAK6 targets to cell-cell adhesions via its N-terminus in a Cdc42-dependent manner to drive epithelial colony escape

PAK6 targets to cell-cell adhesions via its N-terminus in a Cdc42-dependent manner to drive epithelial colony escape

PDCD10 (CCM3) regulates brain endothelial barrier integrity in cerebral cavernous malformation type 3: role of CCM3-ERK1/2-cortactin cross-talk

The focal adhesion targeting domain of p130Cas confers a mechanosensing function

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