Abstract
Cerebral cavernous malformation (CCM) is a vascular malformation of the central nervous system that is associated with leaky capillaries, and a predisposition to serious clinical conditions including intracerebral hemorrhage and seizures. Germline or sporadic mutations in the CCM1/KRIT1 gene are responsible for the majority of cases of CCM. In this article, we describe the original characterization of the CCM1/KRIT1 gene. This cloning was done through the use of a variant of the yeast two-hybrid screen known as the interaction trap, using the RAS-family GTPase KREV1/RAP1A as a bait. The partial clone of KRIT1 (Krev1 Interaction Trapped) initially identified was extended through 5′RACE and computational analysis to obtain a full-length cDNA, then used in a sequential screen to define the integrin-associated ICAP1 protein as a KRIT1 partner protein. We discuss how these interactions are relevant to the current understanding of KRIT1/CCM1 biology, and provide a protocol for library screening with the Interaction Trap.
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Bacigaluppi S, Retta SF, Pileggi S et al (2013) Genetic and cellular basis of cerebral cavernous malformations: implications for clinical management. Clin Genet 83:7–14
Labauge P, Denier C, Bergametti F et al (2007) Genetics of cavernous angiomas. Lancet Neurol 6:237–244
Craig HD, Gunel M, Cepeda O et al (1998) Multilocus linkage identifies two new loci for a mendelian form of stroke, cerebral cavernous malformation, at 7p15-13 and 3q25.2-27. Hum Mol Genet 7:1851–1858
Denier C, Labauge P, Brunereau L et al (2004) Clinical features of cerebral cavernous malformations patients with KRIT1 mutations. Ann Neurol 55:213–220
Serebriiskii I, Estojak J, Sonoda G et al (1997) Association of Krev-1/rap1a with Krit1, a novel ankyrin repeat-containing protein encoded by a gene mapping to 7q21-22. Oncogene 15:1043–1049
Kitayama H, Sugimoto Y, Matsuzaki T et al (1989) A ras-related gene with transformation suppressor activity. Cell 56:77–84
Nassar N, Horn G, Herrmann C et al (1995) The 2.2 A crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf1 in complex with Rap1A and a GTP analogue. Nature 375:554–560
Herrmann C, Horn G, Spaargaren M et al (1996) Differential interaction of the Ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor. J Biol Chem 271:6794–6800
Buss JE, Quilliam LA, Kato K et al (1991) The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein. Mol Cell Biol 11:1523–1530
Jelinek MA, Hassell JA (1992) Reversion of middle T antigen-transformed Rat-2 cells by Krev-1: implications for the role of p21c-ras in polyomavirus-mediated transformation. Oncogene 7:1687–1698
Sato KY, Polakis PG, Haubruck H et al (1994) Analysis of the tumor suppressor activity of the K-rev-1 gene in human tumor cell lines. Cancer Res 54:552–559
Quinn MT, Parkos CA, Walker L et al (1989) Association of a Ras-related protein with cytochrome b of human neutrophils. Nature 342:198–200
Bokoch GM (1995) Regulation of the phagocyte respiratory burst by small GTP-binding proteins. Trends Cell Biol 5:109–113
Eklund EA, Marshall M, Gibbs JB et al (1991) Resolution of a low molecular weight G protein in neutrophil cytosol required for NADPH oxidase activation and reconstitution by recombinant Krev-1 protein. J Biol Chem 266:13964–13970
Irani K, Xia Y, Zweier JL et al (1997) Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science 275:1649–1652
Fashena SJ, Serebriiskii I, Golemis EA (2000) The continued evolution of two-hybrid screening approaches in yeast: how to outwit different preys with different baits. Gene 250:1–14
Brent R, Ptashne M (1985) A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell 43:729–736
Ma J, Ptashne M (1988) Converting a eukaryotic transcriptional inhibitor into an activator. Cell 55:443–446
Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. Nature 340:245–246
Gyuris J, Golemis E, Chertkov H et al (1993) Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75:791–803
Estojak J, Brent R, Golemis EA (1995) Correlation of two-hybrid affinity data with in vitro measurements. Mol Cell Biol 15:5820–5829
Fashena SJ, Serebriiskii IG, Golemis EA (2000) LexA-based two-hybrid systems. Methods Enzymol 328:14–26
Golemis EA, Brent R (1992) Fused protein domains inhibit DNA binding by LexA. Mol Cell Biol 12:3006–3014
Golemis EA, Serebriiskii I, Finley RL Jr et al (2011) Interaction trap/two-hybrid system to identify interacting proteins. Curr Protoc Cell Biol. Chapter 17:Unit 17 13
Golemis EA, Serebriiskii I, Law SF (1999) The yeast two-hybrid system: criteria for detecting physiologically significant protein-protein interactions. Curr Issues Mol Biol 1:31–45
Khazak V, Estojak J, Cho H et al (1998) Analysis of the interaction of the novel RNA polymerase II (pol II) subunit hsRPB4 with its partner hsRPB7 and with pol II. Mol Cell Biol 18:1935–1945
Khazak V, Golemis EA, Weber L (2005) Development of a yeast two-hybrid screen for selection of human Ras-Raf protein interaction inhibitors. Methods Mol Biol 310:253–271
Serebriiskii I, Estojak J, Berman M et al (2000) Approaches to detecting false positives in yeast two-hybrid systems. BioTechniques 28(328–330):332–326
Serebriiskii I, Khazak V, Golemis EA (1999) A two-hybrid dual bait system to discriminate specificity of protein interactions. J Biol Chem 274:17080–17087
Serebriiskii IG, Mitina O, Pugacheva EN et al (2002) Detection of peptides, proteins, and drugs that selectively interact with protein targets. Genome Res 12:1785–1791
Toby GG, Golemis EA (2001) Using the yeast interaction trap and other two-hybrid-based approaches to study protein-protein interactions. Methods 24:201–217
Zhang Y, Lindblom T, Chang A et al (2000) Evidence that dim1 associates with proteins involved in pre-mRNA splicing, and delineation of residues essential for dim1 interactions with hnRNP F and Npw38/PQBP-1. Gene 257:33–43
Finley RL Jr, Brent R (1994) Interaction mating reveals binary and ternary connections between Drosophila cell cycle regulators. Proc Natl Acad Sci U S A 91:12980–12984
Eerola I, Plate KH, Spiegel R et al (2000) KRIT1 is mutated in hyperkeratotic cutaneous capillary-venous malformation associated with cerebral capillary malformation. Hum Mol Genet 9:1351–1355
Laberge-Le Couteulx S, Jung HH, Labauge P et al (1999) Truncating mutations in CCM1, encoding KRIT1, cause hereditary cavernous angiomas. Nat Genet 23:189–193
Sahoo T, Johnson EW, Thomas JW et al (1999) Mutations in the gene encoding KRIT1, a Krev-1/rap1a binding protein, cause cerebral cavernous malformations (CCM1). Hum Mol Genet 8:2325–2333
Zhang J, Clatterbuck RE, Rigamonti D et al (2000) Mutations in KRIT1 in familial cerebral cavernous malformations. Neurosurgery 46:1272–1277. discussion 1277-1279
Eerola I, Mcintyre B, Vikkula M (2001) Identification of eight novel 5′-exons in cerebral capillary malformation gene-1 (CCM1) encoding KRIT1. Biochim Biophys Acta 1517:464–467
Sahoo T, Goenaga-Diaz E, Serebriiskii IG et al (2001) Computational and experimental analyses reveal previously undetected coding exons of the KRIT1 (CCM1) gene. Genomics 71:123–126
Zhang J, Clatterbuck RE, Rigamonti D et al (2000) Cloning of the murine Krit1 cDNA reveals novel mammalian 5′ coding exons. Genomics 70:392–395
Liu W, Draheim KM, Zhang R et al (2013) Mechanism for KRIT1 release of ICAP1-mediated suppression of integrin activation. Mol Cell 49:719–729
Zawistowski JS, Serebriiskii IG, Lee MF et al (2002) KRIT1 association with the integrin-binding protein ICAP-1: a new direction in the elucidation of cerebral cavernous malformations (CCM1) pathogenesis. Hum Mol Genet 11:389–396
Chang DD, Wong C, Smith H et al (1997) ICAP-1, a novel beta1 integrin cytoplasmic domain-associated protein, binds to a conserved and functionally important NPXY sequence motif of beta1 integrin. J Cell Biol 138:1149–1157
Zhang J, Clatterbuck RE, Rigamonti D et al (2001) Interaction between krit1 and icap1alpha infers perturbation of integrin beta1-mediated angiogenesis in the pathogenesis of cerebral cavernous malformation. Hum Mol Genet 10:2953–2960
Zawistowski JS, Stalheim L, Uhlik MT et al (2005) CCM1 and CCM2 protein interactions in cell signaling: implications for cerebral cavernous malformations pathogenesis. Hum Mol Genet 14:2521–2531
Uhlik MT, Abell AN, Johnson NL et al (2003) Rac-MEKK3-MKK3 scaffolding for p38 MAPK activation during hyperosmotic shock. Nat Cell Biol 5:1104–1110
Borikova AL, Dibble CF, Sciaky N et al (2010) Rho kinase inhibition rescues the endothelial cell cerebral cavernous malformation phenotype. J Biol Chem 285:11760–11764
Kleaveland B, Zheng X, Liu JJ et al (2009) Regulation of cardiovascular development and integrity by the heart of glass-cerebral cavernous malformation protein pathway. Nat Med 15:169–176
Zheng X, Riant F, Bergametti F et al (2014) Cerebral cavernous malformations arise independent of the heart of glass receptor. Stroke 45:1505–1509
Gunel M, Laurans MS, Shin D et al (2002) KRIT1, a gene mutated in cerebral cavernous malformation, encodes a microtubule-associated protein. Proc Natl Acad Sci U S A 99:10677–10682
Beraud-Dufour S, Gautier R, Albiges-Rizo C et al (2007) Krit 1 interactions with microtubules and membranes are regulated by Rap1 and integrin cytoplasmic domain associated protein-1. FEBS J 274:5518–5532
Liu JJ, Stockton RA, Gingras AR et al (2011) A mechanism of Rap1-induced stabilization of endothelial cell–cell junctions. Mol Biol Cell 22:2509–2519
Whitehead KJ, Chan AC, Navankasattusas S et al (2009) The cerebral cavernous malformation signaling pathway promotes vascular integrity via Rho GTPases. Nat Med 15:177–184
Maddaluno L, Rudini N, Cuttano R et al (2013) EndMT contributes to the onset and progression of cerebral cavernous malformations. Nature 498:492–496
Cuttano R, Rudini N, Bravi L et al (2016) KLF4 is a key determinant in the development and progression of cerebral cavernous malformations. EMBO Mol Med 8:6–24
Goitre L, De Luca E, Braggion S et al (2014) KRIT1 loss of function causes a ROS-dependent upregulation of c-Jun. Free Radic Biol Med 68:134–147
Goitre L, Distefano PV, Moglia A et al (2017) Up-regulation of NADPH oxidase-mediated redox signaling contributes to the loss of barrier function in KRIT1 deficient endothelium. Sci Rep 7:8296
Antognelli C, Trapani E, Delle Monache S et al (2018) KRIT1 loss-of-function induces a chronic Nrf2-mediated adaptive homeostasis that sensitizes cells to oxidative stress: Implication for Cerebral Cavernous Malformation disease. Free Radic Biol Med 115:202–218
Duttweiler HM (1996) A highly sensitive and non-lethal beta-galactosidase plate assay for yeast. Trends Genet 12:340–341
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Serebriiskii, I.G., Elmekawy, M., Golemis, E.A. (2020). Identification of the KRIT1 Protein by LexA-Based Yeast Two-Hybrid System. In: Trabalzini, L., Finetti, F., Retta, S. (eds) Cerebral Cavernous Malformations (CCM) . Methods in Molecular Biology, vol 2152. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0640-7_20
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DOI: https://doi.org/10.1007/978-1-0716-0640-7_20
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