BCCIP associates with the receptor protein tyrosine phosphatase PTPmu.

Protein tyrosine phosphorylation is recognized as an important mode of regulation for many cellular functions. The tyrosine phosphorylation state of cellular proteins is determined by the coordinated activity of protein tyrosine kinases (PTKs) [Machida et al., 2003; Pawson, 2002] and protein tyrosine phosphatases (PTPs)[Tonks, 2006]. PTPs are a diverse group of enyzmes that exist as either soluble cytoplasmic forms or as transmembrane, receptor-like proteins [Tonks, 2006]. In this manuscript, we focus on the receptor protein tyrosine phosphatase (RPTP) PTPμ. The extracellular segment of PTPμ contains sequences found in several cell adhesion molecules including a MAM domain, an Ig domain and four FNIII repeats. As suggested by the presence of these domains, PTPμ mediates cell-cell adhesion via homophilic binding [Brady-Kalnay et al., 1993; Gebbink et al., 1993b] meaning the “ligand” for PTPμ is an identical PTPμ molecule on an adjacent cell. The juxtamembrane domain of PTPμ contains a region homologous to the conserved intracellular domain of the cadherins. Cadherins are calcium-dependent cell-cell adhesion molecules that associate with the actin cytoskeleton via catenins [Gumbiner, 2005; Lilien, 2005]. Members of the catenin family bind the intracellular domain of cadherins and include α-catenin, β-catenin, plakoglobin and p120. PTPμ associates with cadherin-catenin complexes [Brady-Kalnay et al., 1998; Brady-Kalnay et al., 1995; Hiscox and Jiang, 1998; Sui et al., 2005]. Specifically, PTPμ interacts with classical cadherins such as N-cadherin, E-cadherin and R-cadherin [Brady-Kalnay et al., 1998] and VE-cadherin [Sui et al., 2005]. PTPμ has been shown also to interact with p120 catenin [Zondag et al., 2000]. The intracellular domain of PTPμ contains two conserved catalytic domains. Of these two phosphatase domains, only the membrane proximal domain possesses catalytic activity [Gebbink et al., 1993a]. PTPμ has been implicated previously in the regulation of E-cadherin-mediated cell-cell adhesion [Hellberg et al., 2002] and both N-cadherin and E-cadherin-mediated neurite outgrowth [Burden-Gulley and Brady-Kalnay, 1999; Oblander et al., 2007]. Furthermore, PTPμ has been shown to interact directly with IQGAP1 [Phillips-Mason et al., 2006], a protein involved in the regulation of cadherin-mediated cell-cell adhesion [Kuroda et al., 1998; Li et al., 1999]. In addition to PTPμ’s role in the regulation of cadherin function, PTPμ appears to either directly or indirectly regulate protein kinase C. In support of this, PTPμ was found to interact with the scaffolding protein RACK1 (receptor for activated C-kinase) in a yeast two-hybrid screen [Mourton et al., 2001], and both PTPμ-mediated neurite outgrowth and PTPμ-mediated axon guidance of retinal ganglion cells require PKCδ activity [Ensslen and Brady-Kalnay, 2004; Rosdahl et al., 2002]. In an effort to elucidate PTPμ-mediated signaling events, we set out to identify PTPμ-interacting proteins using a modified yeast two-hybrid screen in which we co-expressed PTPμ with a constitutively active Src tyrosine kinase. The presence of active Src increased the potential to identify proteins that interact in response to tyrosine phosphorylation. In this manuscript, we describe BCCIP as a PTPμ-interacting protein isolated by this modified two-hybrid screen. BCCIP was originally described as TOK-1 (twenty-one and CDK associated protein-1) based on its ability to bind the cyclin-dependent kinase (CDK)- inhibitor, p21Waf1/Cip1 (referred to as p21 hereafter) in a cyclin/CDK2/p21 ternary complex and to enhance the inhibitory activity of p21 on CDK2 [Ono et al., 2000]. Independently, a second group identified BCCIP as a protein that interacts with the internal conserved region of BRCA2, and showed that overexpression of BCCIP inhibits the growth of some tumor cell lines [Liu et al., 2001]. TOK-1 and BCCIP are identical proteins existing as two isoforms in humans, BCCIPα and BCCIPβ [Liu et al., 2001; Ono et al., 2000]. In addition to interacting with p21, BCCIP has been shown recently to regulate p21 expression in a p53-dependent manner [Meng et al., 2004b]. In lower organisms, including rodents, only one BCCIP isoform exists [Lu et al., 2005]. A yeast homolog of BCCIP, Bcp1 has been identified and demonstrated to function in the nuclear export of a protein involved in the regulation of the yeast PI4P 5-kinase, Mss4 [Audhya and Emr, 2003]. In this manuscript, we demonstrate a direct interaction between PTPμ and BCCIP. Furthermore, we show BCCIPα is tyrosine phosphorylated in vitro and a substrate for PTPμ. The best characterized biological function of PTPμ is neurite outgrowth. The catalytic activity of PTPμ is required for it to promote neurite outgrowth of nasal retinal ganglion cells and induce repulsion of temporal retinal ganglion cells [Ensslen-Craig and Brady-Kalnay, 2005]. In this manuscript, we demonstrate that expression of a BCCIP antisense construct blocks both permissive and repulsive activities of PTPμ in neurite outgrowth assays of retinal ganglion cells, suggesting BCCIP and PTPμ are in a common signaling pathway.