©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CD45 Protein-tyrosine Phosphatase Associates with the WW Domain-containing Protein, CD45AP, through the Transmembrane Region (*)

(Received for publication, June 2, 1995; and in revised form, September 1, 1995)

Ellen D. Cahir McFarland (§) Matthew L. Thomas (¶)

From the Howard Hughes Medical Institute, the Departments of Pathology and Molecular Microbiology and the Center for Immunology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

CD45 is a transmembrane protein-tyrosine phosphatase required for antigen receptor signaling in lymphocytes. CD45 activates the Src family protein-tyrosine kinases, p56and p59, by dephosphorylating a negative regulatory tyrosine in the carboxyl terminus. Immunoprecipitation of CD45 precipitates p56 and CD45AP. Although the function of CD45AP is unknown, it has been proposed to be an adapter between p56 and CD45. To assess the ability of CD45AP to function as an adapter, we determined the regions required for the interaction with CD45 by expressing chimeric proteins in HeLa cells. CD45AP has a region similar to a potential protein-protein interaction domain, the WW domain. Surprisingly, this domain was not necessary for the association with CD45. Rather, a 40-amino acid sequence encompassing the putative transmembrane domain of CD45AP was sufficient to mediate binding to CD45. Similarly, a 39-amino acid sequence encompassing the CD45 transmembrane region was sufficient to direct the interaction with CD45AP. Expression of p56 with CD45AP resulted in an interaction that could only be detected by in vitro kinase reaction, suggesting that the association of p56 and CD45AP is weak. These data support a model in which CD45AP links CD45 with other proteins but not necessarily p56.


INTRODUCTION

Engagement of the antigen receptor on T cells results in the activation of protein-tyrosine kinases coupling antigen recognition to subsequent signaling events. Two members of the Src family of protein-tyrosine kinases, p56 and p59, have been implicated in T cell receptor (TcR) (^1)signaling. Therefore, protein-tyrosine phosphatases are predicted to be antagonistic to receptor-signaling events. However, CD45, the major transmembrane protein-tyrosine phosphatase expressed by leukocytes, is required for signaling in T cells. Both CD4 and CD8 CD45-deficient T cell clones are impaired in the ability to proliferate to antigen(1, 2) . Furthermore, CD45-deficient T cell leukemic lines fail to induce tyrosine phosphorylation in response to CD3 cross-linking(3) . Thus, CD45 is required to initiate the earliest steps of T cell receptor signaling.

p56 and p59 from CD45-deficient cells are increased in tyrosine phosphorylation, suggesting that both enzymes are substrates of CD45. All Src family members are negatively regulated by tyrosine phosphorylation(4, 5, 6) . Peptide mapping indicates that the site of increased phosphorylation is the negative regulatory site. Consequently, both kinases from CD45-deficient cells are decreased in kinase activity(6) . The difference in tyrosine phosphorylation and kinase activity is observed when the T cell clones are extensively rested, suggesting that the requirement for CD45 is prior to antigen receptor engagement(6) . Therefore, it is likely that the defect in CD45-deficient cells is the failure to activate the Src family kinases prior to antigen receptor engagement.

Comparison of p56 and p59 from CD45-deficient and -expressing L3 T cells demonstrates an 8- and 2-fold increase in tyrosine phosphorylation, respectively(6) . In the L3 T cell clone, p59 is expressed at nearly twice the level of p56, (^2)yet both are comparable substrates for CD45 in vitro. Therefore, it appears that CD45 preferentially interacts with p56. Furthermore, a chimeric protein consisting of the amino-terminal unique domain of p59 and the remaining carboxyl-terminal portion of p56 is phosphorylated at the negative regulatory site in a manner similar to p59(7) . Interestingly, p60 does not demonstrate increased phosphorylation at the negative regulatory site if transfected into either CD45-expressing or -deficient cells(5) . Therefore, the interaction of CD45 with distinct Src family members appears to be regulated. It is possible additional proteins, which interact with CD45 may modulate substrate accessibility.

A direct interaction between p56 and CD45 has been demonstrated upon phosphorylation of CD45 by p50in vitro(8) . Purified p56 SH2 domains added in excess can inhibit the binding of p56 to CD45. However, it is unclear as to whether this is the mechanism of interaction in vivo.

In vitro kinase reactions of CD45 immunoprecipitates demonstrate the presence of p56 and an associated protein of M(r) 36,000, CD45AP, also termed lymphocyte phosphatase-associated protein(9, 10, 11) . Although CD45 is expressed by all nucleated cells of hematopoietic origin, CD45AP expression is restricted to lymphocytes(11) . The molecular mechanism by which CD45 and CD45AP associate are unknown. Furthermore, it is unknown whether CD45AP is required for the association of CD45 and p56.

Sequence analysis of the mouse and human CD45AP cDNAs predicts a mature protein of 198 amino acids containing a 20-amino acid leader sequence, a 7-amino acid extracellular domain, a membrane-spanning region of 21 amino acids, and a 150-amino acid cytoplasmic domain (10, 11, 12) . Interestingly, the first 50 amino acids of the cytoplasmic domain of CD45AP have significant sequence similarity to a potential protein interaction domain, the WW domain (Fig. 1). The signature residues of a WW domain are highly conserved tryptophan residues. WW domains are found in proteins with diverse functions (13, 14) including dystrophin, utrophin, Ess 1, a protein required for cell division in yeast, and Yap, a protein that associates with p60. It is possible WW domains may function in a manner similar to SH2, SH3, or PH domains(14) .


Figure 1: Comparison of a potential WW domain within CD45AP with other putative WW domains. Sequences are derived from (14) . Identical residues and conservative replacements found in the majority of the sequences are indicated by boldface.



To define the sites by which CD45 and CD45AP interact, we expressed chimeric proteins between CD45 and CD8 or CD45AP and p60. These studies demonstrate that CD45 and CD45 interact through their respective transmembrane regions. Furthermore, co-immunoprecipitations studies suggest that the association between CD45 and CD45AP is much stronger than the association between CD45AP and p56.


EXPERIMENTAL PROCEDURES

Antibodies

Anti-CD8 monoclonal antibody, 53-6.72, was obtained from the ATCC (Rockville, MD) or purchased from Pharmigen (San Diego, CA). Anti-CD45 monoclonal antibody, I3/2.3, was obtained from Dr. Ian Trowbridge (Salk Institute, San Diego, CA). Anti-CD45 antiserum was generated to recombinant cytoplasmic domain of CD45. Anti-c-myc monoclonal antibody, 9E10, was obtained from the ATCC (CRL 1729) and recognizes a specific epitope contained within the sequence SMEQKLISEEDLNN. Anti-ICAM-1 monoclonal antibody CL 203 was obtained from Dr. Michael Dustin (Washington University, St. Louis, MO). Anti-p56 monoclonal antibody 3A5 was obtain from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-p60 monoclonal antibody 327 was obtained from Oncogene Science (Manhasset, NY). Anti-p56 antiserum was made to the peptide EVRDPLVYEGSLPPASPLQDN.

cDNA Reagents

Mouse CD45 cDNA has been described previously(15) . cDNAs for p56 and p60 were provided by Dr. Andrey Shaw (Washington University, St. Louis MO). CD8alpha cDNA was a gift from Dr. Hiromitsu Nakauchi (RIKEN, Tsukuba-city, Japan). To isolate CD45AP cDNA, RNA was isolated from a T cell hybridoma, L3H. Reverse transcriptase polymerase chain reactions were performed with 5 µg of L3H RNA in a cDNA cycle kit (Invitrogen, San Diego, CA) according to the manufacture's instructions. For the polymerase chain reaction, the 5`-oligonucleotide was TCTAGATTGAATTCCACCATGGCCCTGCCAGGG and contained the initiator methionine. The oligonucleotide was modified from the published sequence (10) to contain a consensus translational initiation site. The 3`-oligonucleotide, GGATCCTAGTGCAGTGACCCG, was derived from the end of the coding region. However, the translational stop codon was modified to glycine. Both the 5`- and 3`-oligonucleotides contained restriction endonuclease sites to facilitate cloning. The derived product was subcloned into the Bluescript vector (Stratagene, La Jolla, CA), which was modified to contain the epitope tag derived from c-myc and translational stop codon. The cDNA was sequenced in its entirety and differed from the published sequence by encoding an arginine for cysteine at amino acid position 79. This sequence is referred to as CD45AP(1). CD45AP(1) was modified to contain the additional sequence GGCCGCGTCGACACCACCATGGCTCTGCCTGGTACCCTCAGATTTCCCCTCCT reported by Shimizu et al.(12) . This sequence contains an additional 12 amino acids and predicts a further 5`-translational initiation site. This cDNA is referred to as CD45AP(2). CD45AP is numbered according to Shimizu et al.(12) .

Construction of Chimeric cDNAs

A panel of chimeric cDNA were generated by using polymerase chain reaction products derived by site overlap extention (Fig. 2). Each polymerase chain reaction product contains unique restriction endonuclease sites such that they could be cloned in the correct direction in the appropriate vectors. All cDNAs were cloned in Bluescript behind the T7 promoter.


Figure 2: Schematic diagram of the chimeric proteins expressed in HeLa cells. Numbers above the bars indicate the position of the amino acids derived from each protein. Portions derived from each protein are indicated as follows: CD45, open bar; CD8, black bar; CD45AP, stippled bar; the epitope-tag derived from c-myc, cross-hatched bar; p60, checkered bar. The dark shading at the amino terminus of CD45AP(2) indicates the 12 potential amino acids encoded by the possible alternatively spliced isoform (see ``Experimental Procedures'').



Expression of Chimeric Proteins

For transfection, HeLa cells were plated at 5 times 10^5 cell/well in 6-well plates for 24 h. The cells were washed twice with Dulbecco's modified Eagle's medium containing penicillin, streptomycin, and 1 mM sodium pyruvate. Recombinant vaccinia virus expressing the T7 RNA polymerase (16) was added at a multiplicity of infection of 10 for 45 min at 37 °C in 1 ml of Dulbecco's modified Eagle's medium. A maximum amount of 5 µg of plasmids was resuspended in a volume of 500 µl of Dulbecco's modified Eagle's medium, vigorously mixed, and 15 µl of LipofectACE (Life Technologies, Inc.) was added. The DNA mixture was added dropwise to the cells. After incubation of 12-14 h, the cells were resuspended in lysis buffer (0.9% Brij 58, 0.1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM Na(3)VO(4), 10 mM NaF, 50 µg/ml aprotinin (Sigma)). Alternately, cells were removed from the plate by treating with phosphate-buffered saline (PBS) containing 50 mM EDTA. A portion was used for flow cytometry analysis, and the remainder was resuspended in lysis buffer.

Immunoprecipitation

One ml of lysate was precleared with 10 µl of a 10% solution of Pansorbin cells (Calbiochem, La Jolla CA), centrifuged at 10,000 rpm for 5 min at 4 °C. The supernatant was divided into two 450-µl portions. Appropriate antibodies, 1 µl, were added to each portion with 30 µl of a 50% slurry of either protein G- or protein A-Sepharose. Lysates were rotated at 4 °C for 2 h, washed once with lysis buffer and 20 µl of SDS-polyacrylamide gel electrophoresis sample buffer containing 2-mercaptoethanol was added.

Western Blot Analysis

Immunoprecipitates were resolved on either a 10 or 12% SDS-polyacrylamide gel, transferred to nitrocellulose, and blocked in PBS containing 0.05% Tween 20 (PBS-T), 2% bovine serum albumin, and 1% nonfat dry milk. Antibodies were used at a 1:500 dilution in PBS-T and incubated with the membranes for 1 h. Following the primary antibody, the membranes were washed 3 times with PBS-T for 5 min and developed with either horseradish peroxidase-conjugated protein A or anti-mouse IgG for 30 min and then washed 3 times with PBS-T. Proteins were visualized using the Enhanced Chemiluminescence Kit (Amersham Corp.).

Kinase Reactions

Immune complexes were washed once with lysis buffer and once with 1 ml of kinase reaction buffer (20 mM Tris-HCl, pH 7.5, 10 mM MnCl(2)). Samples were resuspended in 10 µl of kinase reaction buffer with 10 µCi of [-P]ATP (Amersham Corp.) for 10 min at room temperature. Reactions were stopped by the addition of 10 µl of sample buffer containing 2-mercaptoethanol.

Flow Cytometry

HeLa cells were washed once with 4 ml of staining buffer (PBS containing 0.02% bovine serum albumin, 0.01% NaN(3)) and incubated with 1 µl of fluoroscein-conjugated anti-CD8 antibody for 30 min on ice. Cells were washed once with 4 ml of staining buffer, gravity filtered through a 35-µm cell strainer cap (Falcon, Lincoln Park, NJ), and analyzed on a FACScan (Becton Dickinson, Franklin Lakes, NJ).


RESULTS

CD45-CD45AP Interactions Can Be Reconstituted in Nonlymphoid Cells

To identify the structural basis of the interaction between CD45 and CD45AP, we expressed both proteins in HeLa cells utilizing recombinant vaccinia virus expressing T7 RNA polymerase and transfection of cDNAs behind the T7 promoter. Since antibodies to CD45AP were unavailable, the cDNA was modified 3` of the coding region with a sequence encoding an epitope derived from c-myc. The epitope is recognized by the monoclonal antibody, 9E10.

CD45 and CD45AP(1) were expressed in HeLa cells either individually or together (Fig. 3A). In this system, neither protein required the presence of the other for expression. This is in contrast to a previous report that indicated that CD45AP was undetectable in CD45-deficient Jurkat cells, suggesting that CD45AP required the presence of CD45(11) . The difference may be due to the transient, high level expression obtained with the vaccinia/HeLa system.


Figure 3: CD45 interacts with CD45AP through the transmembrane region. A, CD45 associates with CD45AP in HeLa cells. CD45 (lanes 3-6) and/or CD45AP (lanes 1, 2, 5, and 6) were expressed in HeLa cells. Cells were lysed and immunoprecipitated with either anti-c-myc monoclonal antibody, 9E10 (lanes 1, 3, and 5) or anti-CD45 monoclonal antibody, I3/2.3 (lanes 2, 4, and 6). Immunoprecipitates were resolved on an SDS-polyacrylamide gel and Western blotted with either anti-CD45 antiserum (top panel) or 9E10 (bottom panel). B, identification of CD45 sequences required for the association with CD45AP. CD45AP(2) (lanes 1-10) and chimeric proteins CD8/8/45 (lanes 3 and 4), CD8/45/45 (lanes 5 and 6), CD8/8/* (lanes 7 and 8), and CD8/45/* (lanes 9 and 10) were expressed in HeLa cells. Cells were lysed and immunoprecipitated with either 9E10 (lanes 1, 3, 5, 7, and 9) or anti-CD8 monoclonal antibody 53-6.72 (lanes 2, 4, 6, 8, and 10). Immunoprecipitates were resolved on an SDS-polyacrylamide gel and Western blotted with 9E10. C, cell surface expression of the chimeric proteins expressed in B. Cells were stained with directly fluorescein-conjugated anti-CD8 and analyzed on a FACScan.



Immunoprecipitation of CD45 co-expressed with CD45AP(1) demonstrated that the two proteins associate in HeLa cells (Fig. 3A). Likewise, CD45 will immunoprecipitate with CD45AP(1) using the anti-c-myc monoclonal antibody. Thus, we conclude that the association of CD45 with CD45AP is direct and does not require the presence of other lymphoid-specific proteins.

CD45 Interacts with CD45AP through the Transmembrane Region

To identify the region through which CD45 and CD45AP interact, we expressed a series of chimeric proteins. A chimeric protein consisting of the extracellular domain of CD8 and the transmembrane and cytoplasmic domain of CD45 (CD8/45/45) retained the ability to interact with CD45AP (Fig. 3B). This indicates that the extracellular domain of CD45 is not required for the interaction. However, a chimeric protein in which a 39-amino acid region containing the CD45 transmembrane region was replaced by the corresponding region from CD8 (CD8/8/45) lost the ability to associate with CD45AP (Fig. 3B). This demonstrated the specificity of the interaction and that the CD45 transmembrane region was necessary for the association with CD45AP(1). To determine whether the CD45 transmembrane region was sufficient to interact with CD45AP, we expressed proteins that consisted of the extracellular domain of CD8 and either the CD8 transmembrane region with an 8-amino acid cytoplasmic tail (CD8/8/*) or CD45 transmembrane region with a 14-amino acid cytoplasmic tail (CD8/45/*) (Fig. 3B). Only the chimeric protein containing the CD45 transmembrane region associated with CD45AP(2). This identified a 39-amino acid sequence encompassing the transmembrane region of CD45 that is both necessary and sufficient to interact with CD45AP(2). Furthermore, the extended sequences at the amino terminus of CD45AP(2) did not alter the interaction with CD45.

CD45AP Transmembrane Domain Is Required to Interact with CD45

To determine whether the CD45AP transmembrane domain mediates the interaction with CD45, we expressed a chimeric protein consisting of the first 42 amino acids of CD45AP(1) and amino acids 2-533 of p60 protein-tyrosine kinase (APsrc) with the CD8 extracellular domain/CD45 transmembrane domain chimera (CD8/45/*) (Fig. 4). The APsrc chimeric protein co-immunoprecipitated with the CD8/45/* chimera, demonstrating that a region encompassing the transmembrane domain of CD45AP was sufficient to interact with the 39-amino acid domain of CD45. This indicated that the CD45AP WW domain was not necessary for the interaction with CD45. Significantly, p60 did not interact with the CD8/45/* chimeric protein demonstrating the specificity of the interaction. Furthermore, neither a mutant lacking the first 40 amino acids of CD45AP(1) nor a chimeric protein containing the first 10 amino acids of p60 and amino acids 52-198 of CD45AP(1) associated the with CD45 transmembrane region (data not shown). These data indicate that CD45 and CD45AP associate through their respective transmembrane regions.


Figure 4: The transmembrane region of CD45AP is required for the interaction with CD45. A, identification of CD45AP sequences required to interact with CD45. CD8/45/* (lanes 1, 2, 7, 8, 9, and 10) p60 (lanes 3, 4, 7, and 8), and APsrc (lanes 5, 6, 9, and 10) were expressed in HeLa cells. Cells were lysed and immunoprecipitated with either anti-p60 antibody 327 (lanes 1, 3, 5, 7, and 9), or anti-CD8 monoclonal antibody, 53-6.72 (lanes 2, 4, 6, 8, and 10). Immunoprecipitates were subjected to a kinase reaction in the presence of [-P]ATP, resolved on an SDS-polyacrylamide gel and proteins visualized by autoradiography. B, cell surface expression of the chimeric proteins expressed in A. Cells were stained with directly fluorescein-conjugated anti-CD8 and analyzed on a FACScan.



Association of p56 with CD45AP

p56 is found in immunoprecipitates of CD45. Therefore, we examined the possibility that CD45AP is an adapter protein to link p56 with CD45. To determine whether CD45AP associates with p56, we co-expressed both proteins. Surprisingly, CD45AP could not be detected in immunoprecipitates of p56 by Western blot analysis (Fig. 5A). Similarly, Western blot analysis did not detect the presence of p56 in CD45AP immunoprecipitates. However, if immunoprecipitates were subjected to kinase reactions, the presence of p56 in the CD45AP immunoprecipitates could be detected (Fig. 5, A and B). p56 kinase activity was undetectable in control ICAM-1 immunoprecipitates, demonstrating the specificity of the interaction of CD45AP with p56 (Fig. 5B). We interpret these results to suggest that the association of CD45AP with p56 is weak. Therefore, we tested the possibility that p56 associates with CD45 directly in the HeLa cells. p56 could be detected in CD45 immunoprecipitates by in vitro kinase reactions, but not by Western blot analysis (data not shown). This supports the hypothesis that the interaction of p56 with CD45 requires an adapter since the direct interaction of the two proteins is weak.


Figure 5: Weak interaction between CD45AP and p56. CD45AP(1) (lanes 1, 2, 5, and 6) and p56 (lanes 3-6) were expressed in HeLa cells. The cells were lysed and immunoprecipitated with either anti-c-myc, 9E10 (lanes 1, 3, and 5) or anti-p56 antiserum (lanes 2, 4, and 6). Half of the immunoprecipitate was resolved on an SDS-polyacrylamide gels and Western blotted with either anti-p56 antibody, 3A5 (top panel) or 9E10 (middle panel). The remaining portion of the immunoprecipitate was subjected to a kinase reaction in the presence of [-P]ATP resolved on an SDS-polyacrylamide gel, and proteins were visualized by autoradiography. B, CD45AP(2) and p56 were expressed in HeLa cells. Anti-c-myc (lane 1), anti-p56(lane 2), and anti-ICAM-1 (lane 3) immunoprecipitates were subjected to kinase reactions as in A. Western blots were performed in parallel and were similar to the results of A (data not shown).




DISCUSSION

These studies demonstrate that CD45 and CD45AP associate through a region encompassing their respective transmembrane regions. The transmembrane regions of both CD45 and CD45AP are highly conserved. Human and shark CD45 are 70% identical in the region we identified as sufficient for interaction with CD45AP. (^3)Similarly, human and mouse CD45AP are 79% identical in the region that can direct binding to CD45. The high degree of conservation in these regions suggests that the association of CD45 and CD45AP is of functional importance. One potential function is that CD45AP may link CD45 to other molecules. The identification of a potential WW domain within CD45AP provides a possible mechanism by which CD45AP could function as an adapter protein. It will be of much interest to determine whether this region mediates interactions with other proteins. It is important to note that WW domains are found in another potential adapter molecule, Yap. Yap interacts with the p60 SH3 domain through a proline-rich sequence carboxyl-terminal to the WW domain(17) .

Chimeric proteins utilizing only the cytoplasmic domain of CD45 expressed in CD45-deficient cells are sufficient to rescue inositol trisphosphate turn over and intracellular calcium fluxes in response to TcR stimulation(18, 19, 20) . This implies that CD45AP is not required for the regulation of Src family member kinases, tyrosine phosphorylation, or second messenger production. It is possible that the association of CD45 with CD45AP is necessary to couple CD45 to responses not measured by these assays.

The transmembrane association of CD45 with CD45AP is significant. Association through the transmembrane regions has been demonstrated for receptor complexes, most notably the T cell and B cell antigen receptors. In the case of the TcR, the alpha- and beta-chains associate with the CD3 complex and -chain homodimers through transmembrane regions. Transport to the plasma membrane requires the association of the - or -chains(21) . CD45AP is expressed only by lymphocytes and, therefore, unless there is an analogous molecule in other hematopoietic-derived cells, CD45AP is not required for the transport of CD45. However, it appears that CD45 expression increases the half-life of CD45AP, possibly by affecting transport(11) .

Oligomerization of receptor components is required for the initiation of many signal transduction cascades. The transmembrane regions of receptor tyrosine kinases is required for dimerization and autophosphorylation. An oncogenic form of the neu receptor tyrosine kinase has a point mutation in the transmembrane domain that stabilizes dimer formation and causes constitutive signal transduction (22) . Therefore, the ability of CD45 to associate with CD45AP through transmembrane regions may affect the signal transduction properties.

A chimeric protein consisting of the extracellular and transmembrane regions of the epidermal growth factor receptor and the cytoplasmic domain of CD45 will rescue TcR-mediated signal transduction in CD45-deficient cells(18) . Addition of epidermal growth factor results in the dimerization of the chimeric receptor and blocks TcR-mediated signal transduction. It is possible that dimerization of CD45 will affect the ability of the TcR to function. Furthermore, CD45AP may mediate that effect, although it appears CD45AP can associate with both monomers and dimers of CD45 isolated by sucrose gradients(23) .

Expression of p56 and CD45AP in HeLa results in a small proportion of p56 associated with CD45AP. Significantly, the proportion of p56 associated with CD45AP could only be detected by in vitro kinase reaction. This suggests that the direct interaction of p56 with CD45AP is weak. The weak interaction of p56 with CD45AP in HeLa cells may be the result of a detergent effect or a co-localization to the caveoli, which are found in HeLa cells but not in lymphocytes(24) . p56 is modified in the amino-terminal region by both myristylation and palmitylation and will associate with glycophosphoinositol-linked (GPI-linked) proteins in detergent-insoluble immunoprecipitates. This is dependent on the palmitylation of the amino-terminal cysteines (25, 26, 27) . p60, which is not palmitylated, does not associate with GPI-linked proteins(25, 28) . Preliminary data indicates that the palmitylation of p56 is required to detect an interaction between CD45AP and p56 in HeLa cells. A chimera of the first 10 amino acids of p60 replacing the first 10 amino acids of p56 did not co-immunoprecipitate with CD45AP in an in vitro kinase reaction (data not shown). The association of p56 with GPI-linked proteins may represent a co-localization to a microdomain or an association driven by the use of detergents since co-capping studies do not demonstrate co-localization of a GPI-linked protein, Thy-1, with the detergent-insoluble glycolipid GM(1)(24) . Therefore, the weak interaction of p56 with CD45AP in HeLa cells may result from an association in a microdomain or the dissolution of the membrane in detergents. Consequently, it is important that a functional relationship be established between CD45AP and p56, since the direct interaction is extremely weak. However, CD45AP may function as an adapter for CD45 if additional lymphocyte specific proteins are required as linkers between CD45AP and p56.

It will be important to determine whether CD45AP affects CD45 function. The demonstration that CD45 and CD45AP interact through their respective transmembrane regions suggests a possible mechanism to block CD45 and CD45AP interaction and examine the effects on transport, dimerization, phosphatase activity, and signal transduction.


FOOTNOTES

*
This work was supported by United States Public Heath Service Grant AI 26363. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by United States Public Health Service Training Grant AI 07163.

An investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Box 8118, Dept. of Pathology, Washington University School of Medicine, St. Louis, MO 63110. Tel.: 314-362-8722; Fax: 314-362-8888; mthomas@immunology.wustl.edu.

(^1)
The abbreviations used are: TcR, T cell receptor; PBS, phosphate-buffered saline.

(^2)
M. Olszowy and A. Shaw, personal communication.

(^3)
M. Okumura, R. J. Matthews, B. Robb, P. Bork, and M. L. Thomas, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. Lisa K. Timson Gauen, Michael Olszowy, and Andrey Shaw for providing reagents and technical advice. We also thank Henry I. Chen and Dr. Marius Sudol help in delineating the putative WW domain in CD45AP.


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