Oncogene (2001) 20, 346 ± 357 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Identi®cation of a novel interaction between integrin b1 and 14-3-3b Dong Cho Han1, Luis G Rodriguez1 and Jun-Lin Guan*,1 1Cancer Biology Laboratories, Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, NY 14853, USA Integrins are cell surface receptors for extracellular a particular a and b subunit. Integrin-mediated cell matrix, which play important roles in a variety of adhesion to the ECM plays important roles in a variety biological processes. 14-3-3 proteins are a highly of biological processes including embryonic develop- conserved family of cytoplasmic proteins that associate ment, wound healing and cancer. with several intracellular signaling molecules in regula- Studies have shown that integrins can mediate signal tion of various cellular functions. Here, we report transduction across the plasma membrane (Aplin et al., identi®cation of an interaction between the integrin b1 1998; Clark and Brugge, 1995; Schwartz et al., 1995). cytoplasmic domain and 14-3-3b by using the yeast two- Integrin-mediated cell adhesion has been shown to hybrid screen. Like several other proteins, the integrin b1 stimulate a variety of signaling cascades including cytoplasmic domain associated with 14-3-3b by a non- activation of protein tyrosine kinases such as FAK and phosphoserine mechanism. The 14-3-3b/integrin b1 Src, protein serine/threonine kinases including protein interaction was con®rmed by in vitro binding assays as kinase C, mitogen-activated protein kinases, and c-Jun well as co-precipitation in vivo. Furthermore, we found N-terminal kinase, lipid kinases like PI-3-kinase, that 14-3-3b co-localized with integrin b1 during the cytoplasmic alkalinization and calcium in¯ux. Interest- early stage of cell spreading on ®bronectin, suggesting a ingly, integrins also mediate inside-out signaling where potential role of the 14-3-3b/integrin b1 interaction in integrin anity for extracellular ligands is regulated by the regulation of cell adhesion. Using tetracycline- a variety of signaling pathways triggerred by other cell regulated expression system, we showed that overexpres- surface receptors. Changes both in integrin conforma- sion of 14-3-3b stimulated cell spreading and migration tion and/or clustering have been suggested to play a on ®bronectin but not on poly-L-lysine. However, the role in integrin anity regulation (Loftus and induced expression of 14-3-3b did not aect tyrosine Liddington, 1997; Shattil et al., 1998). phosphorylation of FAK or its substrates, p130cas and The cytoplasmic domains of integrins play a key role paxillin, suggesting that 14-3-3b regulated integrin- in both integrin signaling and anity regulation. mediated cell spreading and migration by FAK-indepen- Despite their relatively small size and lack of enzymatic dent mechanisms. Taken together, these results identify activity, integrin b cytoplasmic domains are sucient an interaction between integrin and 14-3-3 proteins and to mediate integrin localization to focal contacts suggest a potentially novel cellular function for 14-3-3 (LaFlamme et al., 1992; Marcantonio et al., 1990; proteins in the regulation of integrin-mediated cell Reszka et al., 1992) and induce FAK phosphorylation adhesion and signaling events. Oncogene (2001) 20, (Akiyama et al., 1994; Guan et al., 1991; Tahiliani et 346±357. al., 1997). They are also required for ®bronectin matrix assembly (Wu et al., 1995) and cell motility (Pasqualini Keywords: integrins; 14-3-3 proteins; cell migration; and Hemier, 1994). The cytoplasmic domains of both protein-protein interaction integrin a and b subunits have been implicated in the regulation of integrin anity (Hughes et al., 1996; O'Toole et al., 1994; 1990). Introduction The critical role of the cytoplasmic domains in integrin function implicates the importance of cyto- The integrins are a family of cell-surface glycoproteins plasmic proteins that modify or bind to integrin which link the extracellular matrix (ECM) to the cytoplasmic domains. Consistent with integrin localiza- cytoskeleton (Hynes, 1992). Each integrin is a hetero- tion in focal contacts, several cytoskeletal proteins dimer that contains an a and a b subunit with each including talin, a-actinin, ®lamin and paxillin have subunit composed of a large extracellular domain, a been shown to interact with cytoplasmic domain of single transmembrane region, and a short cytoplasmic integrin b subunit, which may mediate integrin- domain (except b4). The speci®city of integrin interac- cytoskeleton connections (Horwitz et al., 1986; Loo et tions with ligands is determined by the combination of al., 1998; Otey et al., 1990; Pfa et al., 1998; Schaller et al., 1995; Sharma et al., 1995). In addition, several intracellular signaling proteins have been reported to associate with integrin b cytoplasmic domains, which *Correspondence: J-L Guan Received 18 July 2000; revised 26 October 2000; accepted 1 play interesting and important regulatory functions. November 2000 These include cytohesion-1 (Kolanus et al., 1996), Integrin b1 interaction with 14-3-3b DC Han et al 347 FAK (Schaller et al., 1995), ILK (Hannigan et al., binding site for clone #11 using yeast two hybrid 1996), b3-endonexin (Shattil et al., 1995), ICAP-1 system (Figure 1). Deletion of 13 amino acids from the (Chang et al., 1997; Zhang and Hemler, 1999), and b1 C-terminus of the integrin b1 cytoplasmic domain Rack1 (Liliental and Chang, 1998). (D4) did not signi®cantly aect its interaction with 14-3-3 proteins are a highly conserved family of clone #11. In contrast, removal of 28 amino acids (D3) dimeric proteins ubiquitously expressed in all eukar- or 42 residues (D1) prevented its binding to clone #11, yotic cells (for review, see Fu et al., 2000; Aitken et al., suggesting that residues between the D3 and D4 1995). Members of 14-3-3 family proteins have been mutants (776 ± 790) were involved in binding to 14-3- shown to bind a number of signaling molecules by 3b. 14-3-3 proteins are adaptor molecules that often both phosphoserine motifs (Muslin et al., 1996; Yae bind to phosphorylated Ser motifs (Yae et al., 1997). et al., 1997) and non-phosphoserine mechanism (Clark The Ser790 residue in the integrin b1 cytoplasmic et al., 1997; Masters et al., 1999; Wang et al., 1999). domain has been reported to be phosphorylated in Many of these 14-3-3-associated proteins are proto- mitotic cell (Hynes, 1992). However, mutation of oncogene and oncogene products. Association of 14-3- Ser790 to Ala did not block the interaction, suggesting 3 proteins with intracellular signaling molecules has that Ser790 was not involved in b1 integrin binding to been proposed to regulate their enzymatic activity, to 14-3-3b. Likewise, mutation of Tyr788 to Phe did not bring dierent binding partners (e.g. enzyme and aect its interaction with 14-3-3b, suggesting that substrate) together, and to stabilize the conformation Tyr788 in the NPXY motif is not necessary for the of the associated proteins. Expression of the 14-3-3 was interaction. Clone #11 also interacted with the increased signi®cantly in lung cancer tissues compared cytoplasmic domain of integrin b3 to a similar level with the neighboring normal part of lung (Nakanishi et as b3-endonexin (Shattil et al., 1995). Together, these al., 1997) and in head and neck squamous carcinoma studies indicated that integrin b1 residues 776 ± 790 (Villaret et al., 2000). 14-3-3g expression was increased (other than Tyr788 or Ser790) that are also conserved in response to vessel damage and proliferative signals in the integrin b3 cytoplasmic domain mediated its (Autieri et al., 1996). These studies suggest that 14-3-3 interaction with 14-3-3b in a phosphorylation indepen- proteins are involved in the regulation of mitogenic dent manner. signaling as well as development of cancer. To further understand integrin signaling and regula- Analysis of integrin b1 interaction with 14-3-3b in vitro tion, we employed the yeast two-hybrid screen to and in mammalian cells identify novel cellular proteins that bind to the integrin b1 cytoplasmic domain. We report here the association To determine whether the 14-3-3b fragment (clone #11, of 14-3-3b with integrin b1 cytoplasmic domain by a encoding residues 30 ± 247) could bind directly to the non-phosphoserine mechanism and the eect of 14-3- integrin b1 cytoplasmic domain in vitro, this fragment 3b expression on integrin-mediated cell spreading and was inserted into the pGEX-2T vector to generate a migration. These results suggest potential role of 14-3-3 GST fusion protein for binding assays. The cytoplas- proteins in the regulation of integrin functions. mic domain of b1 integrin was inserted into a mammalian expression vector pKH3 fused in frame with a triple HA epitope tag and a GCN4 dimerization segment. This plasmid was transfected into CHO cells Results and lysates were prepared and incubated with the GST fusion proteins that had been immobilized on Identification of integrin b1 cytoplasmic domain glutathione-agarose beads. After washing, the bound interaction with 14-3-3b by yeast two hybrid screen We employed yeast two-hybrid screen to identify protein(s) that can interact with the cytoplasmic domain of b1 integrin. A cDNA fragment encoding the complete b1 cytoplasmic domain was inserted into a modi®ed pAS2 vector (see Materials and methods) and used to screen a HeLa cell library (Hannon et al., 1993). From approximately 26106 transformants, four clones (clones #10, #11, #20 and #21) were identi®ed that speci®cally interacted with the bait plasmid. Plasmid DNA from these clones was then isolated. Partial DNA sequencing indicated that clone #11 Figure 1 Interaction of 14-3-3b with b1 integrin cytoplasmic encodes almost a complete cDNA for 14-3-3b missing domain and mutants in yeast two-hybrid system.