Serine Phosphorylation-dependent Association of the Band 4.1-related Protein-tyrosine Phosphatase PTPH1 with 14-3-3β Protein*

PTPH1 is a human protein-tyrosine phosphatase with homology to the band 4.1 superfamily of cytoskeletal-associated proteins. PTPH1 was found to associate with 14-3-3β using a yeast two-hybrid screen, and its interaction could be reconstituted in vitro using recombinant proteins. Examination of the interaction between 14-3-3β and various deletion mutants of PTPH1 by two-hybrid tests suggested that the integrity of the PTP is important for this binding. Although both PTPH1 and Raf-1 form complexes with 14-3-3β, they appear to do so independently. Binding of 14-3-3β to PTPH1in vitro was abolished by pretreating PTPH1 with potato acid phosphatase and was greatly enhanced by pretreating with Cdc25C-associated protein kinase. Thus the association between PTPH1 and 14-3-3β is phosphorylation-dependent. Two novel motifs RSLS359VE and RVDS853EP in PTPH1 were identified as major 14-3-3β-binding sites, both of which are distinct from the consensus binding motif RSXSXP recently found in Raf-1. Mutation of Ser359 and Ser853 to alanine significantly reduced the association between 14-3-3β and PTPH1. Furthermore, association of PTPH1 and 14-3-3β was detected in several cell lines and was regulated in response to extracellular signals. These results raise the possibility that 14-3-3β may function as an adaptor molecule in the regulation of PTPH1 and may provide a link between serine/threonine and tyrosine phosphorylation-dependent signaling pathways.

Protein-tyrosine kinases and protein-tyrosine phosphatases (PTP) 1 are the counterparts that orchestrate many aspects of cellular signaling events during such fundamental processes as cell growth and differentiation (1). PTPH1 (2) and PTPMEG1 (3) are the founding members of an expanding group of PTPs characterized by N-terminal segments of homology to cytoskeletal-associated proteins of the band 4.1 superfamily that in-cludes band 4.1, ezrin, talin, radixin, moesin, and merlin (4). Newly identified members of this group of PTPs include PTP-BAS/1E/L1 (5-7), PTPD1/2E/RL10 (8 -10), and PTP36/PEZ (11,12). In these enzymes, the band 4.1 domain and PTP domain are separated by a central segment that contains one to five (PTPH1 has one) PDZ domains (also known as DHR or GLGF domains) (13). PDZ domains are involved in regulating proteinprotein interactions, as illustrated in the interactions of postsynaptic density protein PSD-95 with neuronal type nitric-oxide synthase N-methyl-D-aspartic acid receptors, and Shaker-type potassium channels (14), PTPBAS (also known as FAP-1) with cell surface receptor FAS (15), and the human homologue of Drosophila discs-large tumor suppressor with band 4.1 protein (16). Band 4.1 homology domains themselves are also responsible for targeting cytoskeletal-associated proteins to cytoskeleton-membrane interfaces (4).
We have previously shown that the N-terminal segment of PTPH1 can regulate its enzymatic function (17). Tryptic removal of the N-terminal segment of PTPH1 activates the enzyme up to 10-fold. The observation of this intramolecular regulation of PTPH1 activity, and the possibility that the regulation of PTPH1 in vivo may be achieved by interaction with multiple proteins through the various binding domains discussed above, stimulated our interest in searching for proteins that interact with PTPH1. As described in the following study, 14-3-3␤ was identified as a PTPH1 interacting protein. 14-3-3s are a family of highly conserved acidic proteins, with molecular masses around 30 kDa, that are involved in cell cycle control, transformation, mitogenic signaling pathways (18 -20), apoptosis (21), and learning (22). Their ability to form homotypic or heterotypic dimers (23) allows 14-3-3 to perform a role as a coordinator, adapter/linker, and scaffold in assembling signaling complexes. Furthermore, crystallographic studies indicate that each monomer in a 14-3-3 dimer contains a large groove that can dock another molecule (24,25). In fact, 14-3-3 proteins have been found to interact with a number of signaling proteins, including Raf (26 -30), polyomavirus MT antigen (31), Bcr and Bcr/Abl (32,33), phosphatidylinositol 3-kinase (34), Cdc25 (35), Cbl (36), tryptophan hydroxylase (37), platelet glycoprotein Ib␣ (38), and protein kinase C (20).
Our initial approach involved a yeast two-hybrid screening strategy. Using full-length PTPH1 as a bait, we have isolated 14-3-3␤ protein from a HeLa cell cDNA library. Stable complexes between PTPH1 and 14-3-3␤ were reconstituted in insect Sf9 cells. We have demonstrated that PTPH1 binds 14-3-3␤ in vitro in a serine phosphorylation-dependent manner. Two motifs in PTPH1 were shown to be involved in 14-3-3␤ binding, one of them is phosphorylated in vitro by C-TAK1 (cdc25C-associated protein kinase). Furthermore, we have shown that PTPH1 and 14-3-3 associate in vivo in several human cell lines and the complex may be regulated in response to extracellular signals. These results raise the possibility that 14-3-3␤ may function as an adaptor molecule in the regulation of PTPH1 and may provide a link between serine/threonine and tyrosine phosphorylation-dependent signaling pathways.

EXPERIMENTAL PROCEDURES
DNA Constructs and Yeast Two-hybrid Screen-Full-length and truncated PTPH1 cDNAs were fused to the GAL4 DNA binding domain in vector pGBT8 to produce baits for yeast two-hybrid screens (see Fig.  1). All constructs were generated by convenient restriction enzyme sites or a polymerase chain reaction. Site-directed mutagenesis was performed using the Muta-Gene kit (Bio-Rad) and confirmed by double strand DNA sequencing. A HeLa cell two-hybrid cDNA library (fused to the GAL4 activation domain) in vector pGADGH and yeast host Saccharomyces cerevisiae strain HF7c were kindly provided by G. Hannon (Cold Spring Harbor Laboratory). Other plasmids used in control experiments were generous gifts of our colleagues at Cold Spring Harbor Laboratory: pGBT8-TCPTP (Y.-F. Hu), pGBT8-lamin (J. Stolarov), and pGADGH-Ste4 (H. Tu). Yeast two-hybrid screening techniques and medium compositions were as described by CLONTECH. Initially, HF7c yeast cells were cotransformed with the full-length PTPH1 construct and the HeLa cDNA library. Interaction between PTPH1 and proteins encoded by the cDNA library will reconstitute the GAL4 activator and activate HIS3 and LacZ reporter genes. HIS3 confers upon yeast the ability to grow on histidine-free medium, whereas LacZ produces ␤-galactosidase that can be detected colorimetrically by filter assays. 3-Aminotriazole was included at 10 mM in all selection media to suppress leaky growth from HIS3.
Antibodies and Immunoblot Analysis-Mouse anti-PTPH1 serum, pAbZ5, and monoclonal antibody, mAbZ2, were generated using purified full-length PTPH1 from Sf9 cells as an antigen (17). Rabbit polyclonal antibodies against 14-3-3␤ (SC#628) and Raf-1 (SC#227) were purchased from Santa Cruz Biotechnology. Monoclonal antibody against Raf-1 (R19120) was purchased from Transduction Laboratories. Enhanced chemiluminescence reagents and secondary antibodies or protein A that were coupled to horseradish peroxidase used in all immunoblot analyses were purchased from Amersham Corp. and used as described previously (17).
Cell Culture, Transfection, in Vivo Labeling, and Immunoprecipitation-Human 293, A431, HaCaT, and Saos-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. Transfection of 293 cells with Raf-1 (kindly provided by L. Van Aelst) and PTPH1 in vector pcDNA3 (Invitrogen) was performed with a standard calcium phosphate method. 32 P labeling of 293 and A431 cells was performed with 0.5 mCi/ml [ 32 P]orthophosphate (ICN) for 9 h in phosphate-free DMEM with 5% fetal bovine serum. In metabolic labeling experiments, 40 h after transfection, cells were labeled for 4 h with 0.1 mCi/ml L-[ 35 S]methionine (DuPont NEN) in methionine-free DMEM supplemented with 5% fetal bovine serum. Sf9 cells were labeled the same way in Grace's medium. Cell lysates were prepared in Nonidet P-40 buffer and clarified as described previously (39). Immunoprecipitation was conducted by incubating cell lysates with antibodies prebound to protein A-Sepharose for 2 h at 4°C. The immune complexes were washed four times with Nonidet P-40 buffer and resolved by SDS-PAGE (9% gels have been used throughout this study), blotted onto Immobilon-P membrane (Millipore), and analyzed by autoradiography and/or immunoblotting. Phosphoamino acid analysis of 32 P-labeled PTPH1 was performed by two-dimensional thin layer chromatography as described elsewhere (40).
Expression and Purification of PTPH1 and 14-3-3␤--PTPH1 was expressed in Sf9 cells and purified by fast protein liquid chromatography as described previously (17). The 14-3-3␤ open reading frame was fused to maltose-binding protein (MBP) in vector pMAL-c2, the fusion proteins (MBP14-3-3␤ and MBP) were expressed in Escherichia coli and purified by binding to amylose resin and eluting with maltose according to the procedure described by the manufacture (New England Biolabs). 14-3-3␤ was also expressed in untagged form in Sf9 cells using a recombinant baculovirus (kindly provided by D. Conklin).
In Vitro Binding and PTP Assays-Purified PTPH1 was incubated with MBP or MBP14-3-3␤ bound on amylose beads in PBST (phosphate-buffered saline with 0.5% Triton X-100) containing 1% bovine serum albumin. After 1 h of incubation at 4°C, the beads were washed four times with PBST. Bound material was then analyzed by anti-PTPH1 immunoblotting. For PTP assays, the binding was performed in PTP buffer (25 mM imidazole, pH 7.2, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin) with the addition of 150 mM NaCl. After washing three times in PTP buffer to remove the NaCl, the bound 14-3-3␤⅐PTPH1 complexes were eluted with 20 mM maltose in PTP buffer. The amount of PTPH1 in these complexes was determined by comparing 14-3-3␤-bound PTPH1 with serial dilutions of known amounts of PTPH1 on an immunoblot. Tyrosine phosphatase activity of PTPH1, either complexed with 14-3-3␤ or uncomplexed, was measured with phosphotyrosyl RCML (reduced, carboxamidomethylated, and maleylated lysozyme), myelin basic protein, and polyGlu:Tyr (4:1) (pGluTyr) as described previously (17,41).
Dephosphorylation of PTPH1-Purified PTPH1 was incubated at 30°C with potato acid phosphatase type VII (PAP, Sigma), at a ratio of 1 g of PTPH1/0.1 unit of PAP, in PAP buffer (20 mM MES-OH, pH 5.5, 1 mM MgCl 2 , 0.1 mM dithiothreitol, 10 g/ml leupeptin, 10 g/ml aprotinin, 10 g/ml lima bean trypsin inhibitor, and 1 mM benzamidine). At various time points, an aliquot (1 g of PTPH1) was taken and processed as described for in vitro binding assays, after addition of 20 mM ␤-glycerol phosphate to inhibit the remaining PAP activity.
Phosphorylation of PTPH1 and Peptide Mapping-Purified PTPH1 was phosphorylated for 30 min at 30°C by recombinant C-TAK1 (42) in the following reaction buffer: 50 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , 2 mM dithiothreitol, 50 M ␥-[ 32 P]ATP (8000 cpm/pmol). For phosphopeptide analysis, 10 g of phosphorylated PTPH1 were purified by SDS-PAGE. The gel slice containing the PTPH1 was then digested overnight in 30 mM NH 4 HCO 3 containing 1 g of sequencing grade trypsin (Boehringer Mannheim). The supernatants containing tryptic peptides were separated on HPLC. The fractions containing the highest radioactive material were processed to determine both amino acid sequence and identity of the phosphorylated residue as described previously (43).

14-3-3␤ Was Isolated as a PTPH1-interacting Protein by
Yeast Two-hybrid Screen-Two hundred positives were isolated from an initial screening of 1 million HeLa cell cDNA clones using full-length PTPH1 as a probe. After subsequent purification and curing processes, plasmids of 30 strong interacting clones were isolated for sequence analysis. Ten of these clones encode 14-3-3␤ fused in-frame with the GAL4 DNA binding domain. As controls under the same conditions, 14-3-3␤ did not interact with another protein-tyrosine phosphatase, TCPTP, or nuclear lamin C (Fig. 1B).
A series of PTPH1 constructs and the 14-3-3␤ clone were cotransformed into yeast to test the specificity of the interaction. Immunoblot analyses of the cotransformed yeast with an anti-GAL4 antibody indicate that each of the PTPH1 constructs was expressed to a similar level (data not shown). As shown in Fig. 1B, PTPH1 interacts specifically with 14-3-3␤, but not with the protein of an unrelated yeast gene Ste4 fused to the activation domain, as indicated by the growth on selection medium without histidine. Furthermore, 14-3-3␤ interacted strongly only with full-length PTPH1 (PTPH1-F) and less strongly with the N-terminal two-thirds of PTPH1 (PTPH1-N). PTPH1-F expressors grew on histidine-free medium containing 30 mM 3-aminotriazole, whereas PTPH1-N expressors grew only on medium containing less than 10 mM 3-aminotriazole. However, 14-3-3␤ did not interact with the A or C domain alone (PTPH1-A and PTPH1-C), nor with full-length PTPH1 containing an internal deletion that removes half of the B domain and half of the C domain (PTPH1-NC, Fig. 1B). We attempted to determine whether the B domain alone interacts with 14-3-3, however, the B domain construct (PTPH1-B) supported growth on histidine-free medium by itself (data not shown). Furthermore, the isolated PDZ motif (PTPH1-PDZ) contained in the B domain did not interact with 14-3-3␤ (data not shown). These results indicate that PTPH1 interacts with 14-3-3␤ specifically in the yeast two-hybrid system and that the integrity of PTPH1 is important for optimal interaction.

Stable Complexes of PTPH1 and 14-3-3␤ Were Reconstituted in Vitro and in Sf9 Cells and Were Detected in Various Human
Cells-To test whether 14-3-3␤ can bind to PTPH1 in vitro, purified PTPH1 from Sf9 cells was incubated with MBP or MBP14-3-3␤ that was prebound to amylose beads. As shown in Fig. 2A, PTPH1 bound specifically to 14-3-3␤, but not the MBP moiety, in a concentration-dependent manner. However, binding of 14-3-3␤ did not result in a significant change in the enzymatic activity of PTPH1, as measured with three different tyrosyl-phosphorylated substrates (RCML, myelin basic protein, and pGluTyr) (Fig. 2B).
To reconstitute the interaction between 14-3-3␤ and PTPH1 in a cellular context, Sf9 cells were coinfected with baculoviruses expressing PTPH1 and 14-3-3␤, and the complexes formed between the two proteins were analyzed by immunoprecipitation. As shown in Fig. 3A, 14-3-3␤ was present in the anti-PTPH1 immune complexes from [ 35 S]methionine-labeled Sf9 cells that were coinfected with PTPH1 and 14-3-3␤ baculoviruses. Similarly, PTPH1 was detected in the anti-14-3-3␤ complexes from the coinfected Sf9 cells. The stoichiometry of the association was estimated to be ϳ2 mol of 14-3-3␤ per mol of PTPH1 by densitometric analysis of Fig. 3A.
To confirm that 14-3-3␤ and PTPH1 associate at physiological levels of expression, anti-14-3-3␤ immune complexes from untransfected human 293, A431, HaCaT, and Saos-2 cells were analyzed. As shown in Fig. 3B, PTPH1 was found in anti-14-3-3␤ immune complexes from each cell line tested. Interestingly, HaCaT cells have more PTPH1 than 293 cells, but less PTPH1 was found in complexes with 14-3-3␤, even though both cell lines have similar amounts of 14-3-3␤ protein. This suggests that the extent of association may be regulated by factors, such as differential phosphorylation or subcellular localization of either protein, in addition to the abundance of the two proteins. Furthermore, treatment of quiescent 293 cells with EGF resulted in a 50% reduction in the association between PTPH1 and 14-3-3␤ (Fig. 3C).
Both PTPH1 and Raf-1 Can Interact with 14-3-3␤ in 293 Cells, but They Are Recovered in Different Complexes-Since both PTPH1 and Raf bind to 14-3-3␤, they both may be recruited into the same multiprotein complexes and function in the same signaling cascades. To address this, immune complexes were analyzed from [ 35 S]methionine-labeled 293 cells that were cotransfected with PTPH1 and Raf-1. As shown in Fig. 4, 35 S-labeled endogenous 14-3-3 proteins were found in both anti-PTPH1 and anti-Raf immune complexes. The significance of the slightly different spectra of 14-3-3 proteins detected in the two immune complexes is currently unknown. Conversely, both PTPH1 and Raf were also detected in anti-14-3-3␤ immune complexes. However, under conditions in which both PTPH1 and Raf were quantitatively immunodepleted from cell lysates by their respective antibodies, PTPH1 was not detected in anti-Raf immune complexes and Raf was PTPH1-PDZ, residues 512-605. B, PTPH1 specifically interacts with 14-3-3␤ in yeast two-hybrid tests. Each sector of the plate was streaked with yeast cotransformed with a bait (PTPH1 segments or the control proteins lamin and TCPTP fused to GAL4 DNA binding domain) and a test protein (14-3-3␤ or the control protein Ste4 fused to GAL4 activation domain). Growth on medium without histidine indicates an interaction of proteins coded by the two plasmids. not observed in anti-PTPH1 immune complexes. Trace amounts of PTPH1 were detected in Raf immune complexes only from 293 cells in which PTPH1 was grossly overexpressed from a stronger promoter (data not shown). This situation is distinct from the association of Bcr and Raf, in which 14-3-3␤ serves as a molecular bridge to direct the interaction (33). This suggests that PTPH1 and Raf do not function simultaneously in the same 14-3-3␤ complexes or signaling cascades.

PTPH1 Is Phosphorylated on Seryl Residues in Both Insect and Mammalian Cells, and Phosphorylation Is Required for the
Association between PTPH1 and 14-3-3␤-In the initial binding assays, we were unable to detect binding between 14-3-3␤ and PTPH1 expressed in E. coli (data not shown). However, using PTPH1 expressed in Sf9 cells the association with 14-3-3␤ was successfully demonstrated in vitro ( Fig. 2A). PTPH1 from A431, 293, and Sf9 cells was phosphorylated on seryl residues (Fig. 5A), and PTPH1 from Sf9 cells contained at least four major phosphorylation sites (data not shown). These findings suggest that eukaryotic modifications such as phosphorylation may play a role in regulating the binding interaction. As indicated in Fig. 5B, the specific interaction of PTPH1 and FIG. 2. 14-3-3␤ binds specifically to PTPH1 in vitro but does not alter enzymatic activity significantly. A, PTPH1 binds to 14-3-3␤ in a concentration-dependent manner. Purified PTPH1 at the indicated concentrations was incubated with MBP14-3-3␤ or MBP bound on amylose beads. The bound material was resolved by SDS-PAGE, stained by Ponceau-S (lower panel), and immunoblotted with anti-PTPH1 (upper panel). Ponceau-S staining indicated the amount of fusion proteins used in the binding, but it was not sensitive enough to detect the PTPH1 bound to the beads. B, binding to 14-3-3␤ did not change PTPH1 enzymatic activity significantly. PTPH1 (39 g) was incubated with MBP14-3-3␤ (100 g) on beads. The bound material was eluted with 200 l of 20 mM maltose in PTP buffer and divided into 10 aliquots. For each substrate, duplicate aliquots were assayed for PTP activity. An equivalent amount of unbound PTPH1 was assayed in parallel. The amount of PTPH1 was determined by comparing immunoblots of 14-3-3␤-bound enzyme with various known amounts of unbound PTPH1 (upper panel). The phosphatase activity of PTPH1 in 14-3-3␤-bound (ϩ) or unbound (Ϫ) was normalized to PTPH1 concentration and is expressed as fold change (lower panel).
C-TAK1 Phosphorylates PTPH1 on a Single Site and Enhances Its Association with 14-3-3␤-To identify specific 14-3-3 binding sites regulated by protein phosphorylation, protein serine/threonine kinases were tested for their ability to phosphorylate PTPH1. C-TAK1 was originally purified as a Cdc25C-associated protein kinase (42). It binds to Cdc25C and phosphorylates serine 216, which represents a perfect 14-3-3 binding motif. 2 As shown in Fig. 6, C-TAK1 phosphorylated PTPH1 on a single site and enhanced 14-3-3␤ binding by Ͼ5fold. When C-TAK1-phosphorylated PTPH1 was trypsinized, only two tryptic peptides (p27 and p28) contained significant 32 P radioactivity. Sequencing of these two peptides indicated that p27 and p28 differ by a single arginine at the N terminus. This is due to the trypsin cleavage at alternative arginines in the amino acid sequence of RRSLS 359 VEH in PTPH1. To identify which serine was phosphorylated by C-TAK1, we subjected p28 to Edman degradation and determined at which cycle a 2 H. Piwnica-Worms, unpublished data.  5. PTPH1 is exclusively phosphorylated on seryl residues and phosphorylation is necessary for the association of PTPH1 and 14-3-3␤. A, phosphoamino acid analysis of PTPH1 from various cells. 32 P-Labeled PTPH1 isolated from the indicated cells was hydrolyzed in 6 M HCl and analyzed by two-dimensional thin layer chromatography and autoradiography. The positions of phosphoserine (P-Ser), phosphothreonine (P-Thr), and phosphotyrosine (P-Tyr) were determined by corresponding standards and are marked by dotted lines. B, dephosphorylation of PTPH1 abolishes its binding to 14-3-3␤. Purified PTPH1 was treated with PAP for the indicated periods and then used in binding assays. The unbound fraction was analyzed in parallel with the bound materials by immunoblotting using anti-PTPH1.
FIG. 6. C-TAK1 phosphorylates PTPH1 on a single site and enhances its association with 14-3-3␤. A, phosphorylation of PTPH1 by C-TAK1 enhances the binding of PTPH1 to 14-3-3␤. PTPH1 was incubated with C-TAK1 in the presence or absence of ATP and then assayed for binding to MBP14-3-3 or MBP by immunoblotting. B, HPLC profile of tryptic peptides from C-TAK1-phosphorylated PTPH1. C-TAK1-phosphorylated PTPH1 was trypsinized and fractionated by HPLC. Radioactivity in each fraction was determined by scintillation counting. C, C-TAK1 phosphorylates PTPH1 on Ser 359 . p28 was sequenced by Edman degradation, and the radioactivity in each cycle was determined and plotted against the amino acid eluted in each cycle. burst of [ 32 P]phosphate was recovered. As shown in Fig. 6C, the third sequencing cycle is the radioactive peak, indicating that Ser 359 is the phosphorylated amino acid.
PTPH1 Contains Novel 14-3-3␤ Binding Motifs Different from the Raf Binding Consensus-Although PTPH1 does not contain an exact copy of the 14-3-3 binding motif corresponding to that found in Raf (RSXSXP) (48), it does have two potential 14-3-3-binding sites (RSLS 359 VEH and RVDS 853 EP) that display some features of the motif. To test whether both RSLS 359 VEH and RVDS 853 EP are involved in binding to 14-3-3␤, serine resides 359 and 853 of PTPH1 were changed to alanine by site-directed mutagenesis (Fig. 7A).
To confirm that 14-3-3␤ binds to Ser 359 , the major site of phosphorylation by C-TAK1, purified Ser 359 to alanine mutant (SA1) was phosphorylated by C-TAK1 and used in binding assays. As demonstrated in Fig. 7B, this mutation diminished the phosphorylation by C-TAK1 by 90% and abolished the enhanced binding to 14-3-3␤.
When lysates from Sf9 cells expressing the two single serine to alanine mutants SA1 and SA2 were incubated with 14-3-3␤ beads, the binding of both mutants to 14-3-3␤ was significantly reduced compared with wild-type PTPH1. The double serine to alanine mutation, SA3, reduced the binding by ϳ80% (Fig. 7C).
Thus in a cellular context phosphorylation of PTPH1 on at least two sites regulates its association with 14-3-3␤. DISCUSSION PTPs have the potential to exert a considerable influence on tyrosine phosphorylation-dependent signaling pathways, both augmenting and antagonizing the function of protein tyrosine kinases. Therefore, it is important to understand the mechanism of regulation of PTP function. Both protein kinases and phosphatases have been shown to be regulated by association with anchor and scaffold proteins (44). In this study, we have demonstrated for the first time an association between PTPH1 and 14-3-3␤ both in vitro and in vivo. Interestingly, both 14-3-3s (20) and PTPH1 are abundantly expressed in brain, 3 and in the rat, PTPH1 is concentrated particularly in thalamus (45).
Since none of the individual domains of PTPH1 alone interacted with 14-3-3␤ in yeast two-hybrid tests, the higher order structure of PTPH1 appears very important for the optimal interaction. We demonstrated further that serine phosphorylation of PTPH1 is a prerequisite for this binding. Binding of 14-3-3␤ to PTPH1 in vitro was abolished by pretreating PTPH1 with PAP and was greatly enhanced by pretreating with C-TAK1. This is in agreement with previous findings of a requirement for phosphorylation in other proteins that interact with 14-3-3 (21,37,46,47). Using in vitro binding assays, we have identified two major 14-3-3␤-binding sites in PTPH1. Of six potential serine phosphorylation sites in PTPH1 with some features of the 14-3-3 consensus recognition site identified in Raf (48), RSLS 359 VEH at the junction of the band 4.1 domain and middle segment and RVDS 853 EP in the catalytic domain display the greatest similarity to the RSXSXP (underline indicates phosphoserine) motif. Mutation of Ser 359 and Ser 853 to alanine reduced the association of phosphatase with 14-3-3␤ in vitro by 80%, indicating that Ser 359 and Ser 853 are the major 14-3-3␤-binding sites. These results indicate that the consensus motif RSXSXP found in Raf is not strictly required for interactions with 14-3-3. Platelet glycoprotein Ib␣ (38), Bcr (32), and tryptophan hydroxylase (37) are known binding proteins of 14-3-3; however, inspection of their sequences also revealed no motif identical to the consensus RSXSXP. Recently, new 14-3-3 binding sequences were found in protooncogene Cbl as RHSLPFS and RLGSTFS (49), which do not fit exactly with the consensus RSXSXP. Taken together, it would appear that the recognition of 14-3-3 can be achieved by multiple proteins containing phosphoserine in motifs that diverge significantly from the one found in Raf.
14-3-3 proteins are a family of at least seven members that display distinct expression patterns in different cells or tissues (20). Similar to the way SH2 domains bind to phosphotyrosyl residues, different isoforms of 14-3-3 proteins may bind specifically to phosphoseryl residues contained in different recognition motifs. The different recognition motifs may be phosphorylated in response to distinct signals, creating an additional level of complexity and specificity in the role of 14-3-3 proteins. There are multiple sites of serine phosphorylation in PTPH1 in vivo (data not shown), including the two sites identified here, raising the possibility that PTPH1 may associate with different spectra of 14-3-3 proteins in response to various signals. Preliminary data suggest that 20% of PTPH1 is associated with 14-3-3␤ protein in quiescent 293 cells (data not shown), whereas this association drops by 50% upon EGF stimulation (Fig. 3C). The differential association of PTPH1 and 14-3-3 in different cell lines may reflect a difference in phosphorylation status or subcellular localization of PTPH1. Through the action Purified-PTPH1 wild-type (WT) and mutant (SA1) were phosphorylated by C-TAK1 and then incubated with MBP and MBP14-3-3␤ bound to beads. Aliquots of the binding mixtures and the bound material were immunoblotted with anti-PTPH1 and subjected to autoradiography. C, mutations of Ser 359 and Ser 853 in PTPH1 significantly reduced their binding to 14-3-3␤. Lysates (2 mg) of Sf9 cells expressing the PTPH1 mutants were incubated with 5 g of MBP or MBP14-3-3␤ bound to beads. Starting lysates (10 g/lane) and bound materials were analyzed by immunoblotting with anti-PTPH1. of 14-3-3, PTPH1 may function in cross-talk between protein serine and tyrosine phosphorylation-dependent steps in signal transduction pathways. We are currently trying to identify proteins that become linked to PTPH1 through binding of 14-3-3.
We have shown previously that the sequences flanking the catalytic domain of PTPH1 have an inhibitory effect on activity (17). However, using artificial substrates we have not been able to detect a significant effect of 14-3-3␤ binding on the phosphatase activity of PTPH1. It is possible that significant effects may be demonstrated with the yet to be identified physiological substrate(s) of PTPH1 instead of the artificial substrates used in the PTP assays to date. Indeed, the direct effects of 14-3-3 binding on the enzymatic properties of associated proteins such as Raf and protein kinase C (20, 26 -30, 47) remain controversial and unclear. It is also possible that significant effects on the PTP activity of PTPH1 may require a combined action of several regulators, such as proteins interacting with the band 4.1 and/or PDZ domain of PTPH1, possibly with 14-3-3␤ being a cofactor.
PTPH1 was phosphorylated exclusively on serine in either insect Sf9 or human 293 and A431 cells. C-TAK1 phosphorylates Ser 359 on PTPH1, one of the two major 14-3-3␤ binding sites, almost exclusively in vitro. Mutation of Ser 359 to alanine reduced by 90% the C-TAK1-mediated phosphorylation of PTPH1 and completely abolished the C-TAK1-mediated enhancement of binding to 14-3-3␤ in vitro. Protein kinase C also phosphorylated PTPH1 and enhanced its binding to 14-3-3␤, but the phosphorylation is complex, involving multiple sites (data not shown). Apart from C-TAK1 and protein kinase C, we have failed to detect significant phosphorylation of PTPH1 and enhancement of 14-3-3␤ binding by protein kinase A and casein kinase II. It is currently unclear which protein kinase phosphorylates PTPH1 in vivo; however, C-TAK1 and members of the protein kinase C family certainly cannot be ruled out.
In conclusion, we have identified 14-3-3␤ as a PTPH1-associated protein. Our findings of 14-3-3␤ binding to novel phosphoserine motifs in PTPH1 further support the notion that protein-protein interaction may play a significant role in the regulation and function of PTPH1, as well as other PTPs, in cross-talk mediated by protein serine and tyrosine phosphorylation cascades.