Journal of Cell Science 112, 3299-3308 (1999) 3299 Printed in Great Britain © The Company of Biologists Limited 1999 JCS0400 A FERM domain governs apical confinement of PTP-BL in epithelial cells Edwin Cuppen, Mietske Wijers, Jan Schepens, Jack Fransen, Bé Wieringa and Wiljan Hendriks* Department of Cell Biology, Institute of Cellular Signalling, University of Nijmegen, Adelbertusplein 1, 6525 EK Nijmegen, The Netherlands *Author for correspondence (e-mail: [email protected]) Accepted 2 July; published on WWW 22 September 1999 SUMMARY PTP-BL is a cytosolic multidomain protein tyrosine nor can we detect a direct interaction by phosphatase that shares homologies with several immunoprecipitation assays. Fluorescence recovery after submembranous and tumor suppressor proteins. Here we photobleaching (FRAP) experiments show that PTP-BL show, by transient expression of modular protein domains confinement is based on a dynamic steady state and that of PTP-BL in epithelial MDCK cells, that the presence of complete redistribution of the protein may occur within 20 a FERM domain in the protein is both necessary and minutes. Our observations suggest that relocation is sufficient for its targeting to the apical side of epithelial mediated via a cytosolic pool, rather than by lateral cells. Furthermore, immuno-electron microscopy on stable movement. Finally, we show that PTP-BL phosphatase expressing MDCK pools, that were obtained using an domains are involved in homotypic interactions, as EGFP-based cell sorting protocol, revealed that FERM demonstrated by yeast two-hybrid assays. Both the highly domain containing fusion proteins are enriched in restricted subcellular compartmentalization and its specific microvilli and have a typical submembranous location associative properties may provide the appropriate at about 10-15 nm from the plasma membrane. conditions for regulating substrate specificity and catalytic Immunofluorescence microscopy suggested colocalization activity of this member of the PTP family. of the FERM domain moiety with the membrane- cytoskeleton linker ezrin. However, at the electron Key words: Polarized, Band 4.1, PDZ, Immuno-electron microscopy, microscopy level this colocalization cannot be confirmed Green fluorescent protein, EGFP, Protein tyrosine phosphatase INTRODUCTION receptor-type RPTPα gives structural support for a model in which enzyme inhibition is the consequence of dimerization, Reversible phosphorylation of proteins on tyrosine residues is by showing that the active site cleft in the subunits is blocked a key event in the regulation of various cellular processes, such by protruding loops of the opposing domain (Bilwes et al., as proliferation, differentiation, cell motility, cell-cell 1996). Structural data on the RPTPµ membrane proximal PTP interactions, metabolism, gene transcription, and the immune domain, however, show that this model might not be response (Hunter, 1996). Proper initiation, propagation and universally applicable (Hoffmann et al., 1997). Also, for the termination of the signaling cascades involved are thus cytosolic PTP SHP-2, for example, it was shown that N- critically dependent on the mode of action of individual terminal SH2 domains are involved in blocking its catalytic site members of the large families of protein tyrosine kinases in an intramolecular interaction (Hof et al., 1998). (PTKs) and protein tyrosine phosphatases (PTPs) (Hunter, Importantly, also targeting of the enzyme to highly restricted 1998; Ninfa and Dixon, 1994). It is now well established that subcellular micro-compartments may control PTP activity and receptor PTKs are activated by an intermolecular mechanism substrate specificity. In this light, the existence of a variety of upon ligand-induced dimerization (Lemmon and Schlessinger, modular protein domains in cytosolic PTPs, that may function 1994). Cytoplasmic PTKs, on the other hand, are activated by as ‘zip codes’ by directing the protein to the correct cellular both intra- and intermolecular mechanisms (Thomas and ‘address’ (Mauro and Dixon, 1994), is intriguing. Candidate Brugge, 1997). Much less is known regarding the processes domains that may regulate activity and specificity are that antagonize the effects of PTKs or, for that matter, the particularly abundant in the large cytosolic protein tyrosine functional activation of PTPs. Recent data suggest that for phosphatase PTP-BL (Hendriks et al., 1995) or RIP (Chida et PTPs similar mechanisms as for PTKs do exist (reviewed by al., 1995), which is the mouse homologue of human PTP- Weiss and Schlessinger, 1998). For example, ligand-induced BAS/PTPL1/PTP1E/FAP-1 (Banville et al., 1994; Maekawa et dimerization of an EGFR-CD45 chimera was found to inhibit al., 1994; Saras et al., 1994; Sato et al., 1995). The PTP activity and TCR signaling (Majeti et al., 1998). The approximately 250 kDa protein, that is expressed mainly in crystal structure of the membrane-proximal PTP domain of the epithelia (Hendriks et al., 1995; Cuppen et al., 1998; Thomas 3300 E. Cuppen and others et al., 1998), harbors a large N-terminal domain with no interaction of the catalytic PTP-BL phosphatase domain. We obvious homology to other proteins except for a potential suggest that subcellular targeting of PTP-BL to a highly leucine zipper (Saras et al., 1994), followed by a FERM restricted microcompartment within the cell, possibly together domain, five PDZ motifs and a C-terminal PTP domain (see with a dimerization-induced shift in activity, may be an also Fig. 2). Outside its natural context, the PTP-BL important determinant in confining its specificity and activity. phosphatase domain has a high rate of activity with a rather promiscuous substrate specificity (Cuppen et al., 1998; Hendriks et al., 1995). For example, a PTP-BL phosphatase MATERIALS AND METHODS domain-GST fusion protein is able to completely eliminate the extensive phosphorylation of endogenous proteins by the Expression plasmid constructions activated tyrosine kinase present in Escherichia coli TKX-1 The cDNA restriction fragments encoding segments of PTP-BL cells (E. Cuppen, unpublished observations). Obviously, a (GenBank accession number Z32740) as indicated in Fig. 2 were regulatory mechanism must be involved in imposing the proper subcloned in pEGFP vectors (Clontech) or in modified versions of the restrictions on PTP-BL’s catalytic activity at endogenous sites. eukaryotic expression vector pSG5 (Green et al., 1988). In the pSG5 The modular domains, like the PDZ and FERM motifs that are vector we have introduced an in-frame VSV-G epitope tag sequence present in PTP-BL, may provide this function in vivo. to allow the production of fusion-proteins that can be detected with the α-VSV monoclonal antibody P5D4. Furthermore, we introduced PDZ motifs are small protein modules that mediate protein- either an initiator AUG codon or a stop codon for proper start or protein interactions (Fanning and Anderson, 1996; termination of translation (see also Cuppen et al., 1998). BL-PDZ-I- Ranganathan and Ross, 1997; Saras and Heldin, 1996) and can V and BL-P were tagged at their N-terminal end, whereas BL-FERM associate with transmembrane proteins (Sheng, 1996), is tagged at its C terminus. A single point mutation at amino acid cytoskeletal components (Xia et al., 1997) and signal 2344, aspartate to alanine, was introduced in BL-P by PCR using transduction enzymes (Ranganathan and Ross, 1997). Indeed, synthetic oligonucleotides, resulting in the catalytically inactive several proteins that interact with PTP-BL PDZ motifs have mutant BL-P-DA. The BL-Nter and the BL-Nter-FERM constructs been identified (Cuppen et al., 1998; Saras et al., 1997; E. were tagged C-terminally with EGFP by subcloning into the pEGFP- Cuppen, unpublished observations), but thus far no clues as to N3 vector (Clontech), whereas BL-FERM+PDZ-I-V and EGFP-BL- explain PTP-BL’s subcellular localization in vivo have been FERM were tagged N-terminally by subcloning into the pEGFP-C1 vector (Clontech). BL-FERM+PDZ-I-V+P was expressed as an obtained. untagged protein from the pSG5 vector. Full length PTP-BL cDNA, The FERM domain in PTP-BL is a segment commonly including its 3′-UTR, was inserted into pEGFP-N3, allowing the found in a family of peripheral membrane proteins that production of untagged PTP-BL (BL-FL). Subsequently, this function as membrane-cytoskeleton linkers (Chishti et al., construct was modified by PCR to generate a PTP-BL-EGFP fusion 1998). This family includes erythrocyte protein 4.1, ezrin, construct (BL-FL-EGFP). To this end, we introduced a XhoI site at radixin and moesin, but also the tumor suppressors Expanded the position of the stop codon, thereby removing the 3′-UTR including and merlin and the protein tyrosine phosphatases PTP-MEG, the stop codon, before introducing the EGFP moiety. Detailed cloning PTPH1, PTPD1 and CDEP. The FERM domain in erythrocyte information can be obtained from the authors upon request. protein 4.1 binds a wide variety of molecules including ATP, Antibodies PIP2, phosphatidylserine, calmodulin, p55, glycophorin A, The affinity-purified polyclonal antisera α-BL-PDZ-I and α-BL-P, glycophorin C, Band 3 and CD44 (see Chishti et al., 1998 for directed against the first PDZ motif and the phosphatase domain of references). Ezrin, radixin and moesin (ERMs) also function PTP-BL, respectively, and the monoclonal α-VSV antibody P5D4 as molecular linkers that connect cell-surface transmembrane have been described elsewhere (Cuppen et al., 1998; Kreis, 1986). The proteins to the