A Novel Macrophage Actin-associated Protein (MAYP) Is Tyrosine-phosphorylated following Colony Stimulating Factor-1 Stimulation*

An ∼37-kDa cytoplasmic protein is rapidly tyrosine-phosphorylated in the response of mouse BAC1.2F5 macrophages to colony stimulating factor-1 (CSF-1). pp37 was purified from the cytosolic fraction by anti-Tyr(P) affinity chromatography, size exclusion chromatography, and C4 reverse phase high pressure liquid chromatography. The sequences of four peptides derived from the purified protein matched portions of an expressed sequence tag (EST) sequence, and the EST clone was used to obtain cDNA clones encoding the pp37 protein, which shares sequence similarity with the PST PIP (proline, serine,threonine phosphatase interactingprotein)/CDC15 family of protein-tyrosine phosphatase substrates. pp37 is predicted to contain a Fes/CIP4 homology (FCH) domain and an actin-binding domain-like sequence. It is expressed selectively in macrophages, macrophage cell lines, and at low levels in macrophage-containing tissues. pp37 is predominantly found in the cytosol, where it is associated with actin. However, ∼4% resides in the membrane fraction, and the trace amount in the cytoskeletal fraction is increased by CSF-1 stimulation. Termedmacrophage actin-associated tyrosine-phosphorylated protein (MAYP), p37 is the major F-actin-associated protein that is tyrosine-phosphorylated in macrophages and is likely to play a role in regulating the CSF-1-induced reorganization of the actin cytoskeleton.

. Incubation of macrophages with CSF-1 causes CSF-1R dimerization, activation, and tyrosine phosphorylation, followed at 1 min after CSF-1 addition by the tyrosine phosphorylation of several, primarily cytoplasmic, proteins, usually associated in complexes with cytoskeletal and/or signaling proteins (4 -9). The tyrosine phosphorylation of the non-CSF-1R proteins may be directly or indirectly mediated by the CSF-1Rkinase or may increase due to growth factor-induced inhibition of a protein-tyrosine phosphatase (PTP).
We have used direct purification and sequencing approaches, to identify several tyrosine-phosphorylated proteins, including the PTP, SHP-1, and Shc, as well as several cytoskeletal and/or signaling molecules associated with them (9,10). In this paper, we describe the characterization of the major macrophage Factin-associated tyrosine-phosphorylated protein (MAYP). This 37-kDa protein shares sequence similarity with a family of PTP substrates, is selectively expressed in macrophages and following CSF-1 stimulation, exhibits increased tyrosine phosphorylation, and is increased in the cytoskeletal fraction.

EXPERIMENTAL PROCEDURES
Cell Culture, Protein Purification, and Sequencing-BAC1.2F5 macrophages (11) were cultured in 100-mm tissue culture dishes and stimulated with 13.2 nM CSF-1 (human recombinant macrophage colony stimulating factor, a gift from Chiron Corp.) at 4 or 37°C, in the presence or in the absence of 8 mM iodoacetic acid (IAA, Fluka), as described previously (9). For purification of pp37, the CSF-1 stimulation of cells for 2 h at 4°C in the presence of IAA to increase the yield of Tyr(P) proteins, their subcellular fractionation, the isolation of the anti-Tyr(P) reactive fraction from the cytosol, its further fractionation by C 4 reverse phase high performance liquid chromatography (RP-HPLC), the endoproteinase-Arg-C (endo-R-C) digestion of selected fractions, and the separation of resulting peptides for microsequencing were performed exactly as described previously (9,10). Cell lines were obtained and bone marrow-derived macrophages (BMM) prepared as described previously (12,13) cDNA Cloning-p37 cDNAs were cloned by screening a previously described ZAPII BAC1.2F5 cDNA library (Stratagene) (12) using a mixture of two 32 P-radiolabeled oligonucleotides 5Ј-gCTggAgAAggTTC-gAgAAgACTggCAgAgTgAACACATTAAggCCTgCg-3Ј and 5Ј-TggA-CAgACTCCCCCAgCACCCATCATgTATgAgAACTTCTACTCTCCTC-3Ј that were based on the expressed sequence tag (EST) vj01 h01.r1 from the Washington University-Howard Hughes Medical Institute mouse EST project. Filters were hybridized in 6 ϫ SSC, 20 mM NaH 2 PO 4 , 0.4% SDS, 500 g/ml salmon sperm DNA at 55°C for 18 h and washed stepwise to a final stringency of 0.5% SCC, 0.1% SDS at 55°C. From the 1.3 ϫ 10 6 phages screened, 36 positive clones were obtained and further analyzed by PCR using a combination of an antisense internal primer (5Ј-TTTgTCTgAATCCTCTACTgC-3Ј) with T3 or T7 primers. The six clones with the longest 5Ј end sequence were recovered in pBlueScriptII SKϪ by phagemid excision. cDNA inserts were characterized by restriction analysis and by sequencing on both strands by the dideoxy nucleotide chain termination method using an automatic sequencer (Applied Biosystems, model 373A). One clone ( 1 The abbreviations used are: CSF-1, colony stimulating factor-1; CSF-1R, CSF-1 receptor; endo-R-C, endoproteinase-Arg-C; EST, expressed sequence tag; FCH, Fes/CIP4 homology; IAA, iodoacetic acid; MAYP, macrophage actin-associated tyrosine-phosphorylated protein; PST PIP, proline, serine, threonine phosphatase interacting protein; Tyr(P), phosphotyrosine; PTP, protein-tyrosine phosphatase; PTP-HSCF, PTP-hematopoietic stem cell fraction; RP-HPLC, reverse phase high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; WASP, Wiskott-Aldrich syndrome protein; BMM, bone marrow-derived macrophages; PCR, polymerase chain reaction; kb, kilobase pair(s); HRP, horseradish peroxidase; ORF, open reading frame; aa, amino acids. h later, cells were extensively washed and transferred into Dulbecco's modified Eagle's medium containing 10% fetal calf serum for another 24 h. Cells were solubilized in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (1% SDS, 50 mM Tris-HCl, pH 6.8, 0.1 M ␤-mercaptoethanol, 6% glycerol, and 0.01% bromphenol blue) and subjected to SDS-PAGE, transfer, and immunoblotting with anti-MAYP antibody.
Two-dimensional Gradient Gel Electrophoresis-The first dimension, isoelectric focusing, utilized a Immobiline DryStrip (Amersham Pharmacia Biotech, pH 3-10, 11 cm) run on a Multiphor II (Amersham Pharmacia Biotech) flat bed electrophoresis tank equipped with Immobiline DryStrip kit (Amersham Pharmacia Biotech). Sample preparation, application, and running conditions of the first dimension isoelectric focusing were carried out as described in the instructions. The gel strips from the first dimension were immersed in SDS-PAGE sample buffer for 2 h at 37°C, without shaking, and then secured with 0.5% agarose in SDS-PAGE sample buffer without glycerol on top of the stacking gel of a 1.5-mm-thick, second dimension SDS-polyacrylamide gradient (7.5-17.5% acrylamide, 2.7% bisacrylamide, Laemmli (15) system) slab gel. The gel was electrophoresed at 8 V/cm for 18 h at 16°C and either silver-stained or transferred to polyvinylidene difluoride membrane for immunoblotting.

Identification of pp37 as a CSF-1-regulated Phosphotyrosyl
Protein-When the anti-Tyr(P)-reactive cytosolic fraction of CSF-1-stimulated BAC1.2F5 macrophages is subjected to SDS-PAGE, pp37 is the major CSF-1-stimulated tyrosine-phosphorylated band of molecular mass Ͻ 50 kDa. Its tyrosine phosphorylation is increased 4 -6-fold by CSF-1 stimulation. If the cells are preincubated with the PTP inhibitor, IAA, p37 is constitutively tyrosine-phosphorylated to ϳ27 times the levels seen in unstimulated cells in the absence of IAA, suggesting that its CSF-1-induced tyrosine phosphorylation could be regulated by inhibition of its dephosphorylation by a PTP (Fig. 3 of Ref. 9). Two-dimensional gel electrophoretic analysis of the anti-Tyr(P)-reactive cytosolic fraction of cells incubated with IAA ( Fig. 1) demonstrates that the pI of tyrosine-phosphorylated pp37 ranges from 6 to 9.5, consistent with its possible phosphorylation on multiple residues (upper and middle panels). This behavior of pp37 was subsequently confirmed by reprobing the immunoblot with anti-MAYP (lower panel).
Purification and Sequence Analysis of pp37-pp37 was purified from cytosol of IAA-treated and CSF-1-stimulated BAC1.2F5 cells by anti-Tyr(P) affinity chromatography, denaturing size exclusion chromatography (9, 10), and C 4 RP-HPLC (Fig. 2). Ten percent of the purified pp37 from the C 4 fraction was used for NH 2 -terminal sequence determination and 90% digested by endo-R-C. The resulting peptides were resolved by C 8 -RP-HPLC and sequenced.
Cloning and the Properties of the Cloned pp37 (MAYP)-A search of the Genbank data base, using the amino acid sequences of nine tryptic peptides derived from purified pp37 and the tblast.n algorithm, yielded a single match with an EST clone. A single ORF in this clone matched four of the nine peptides. Two 50-mer oligonucleotides based on the sequence of that clone were used to screen a ZapII BAC1.2F5 cDNA library. Thirty-six independent hybridizing clones were obtained. The corresponding primary phages were further analyzed by PCR using an EST internal antisense primer and T3 or T7 primers. The six clones containing the longest 5Ј end sequences were rescued and sequenced. The sequence of the larger cDNA insert (2.1 kb) contained a full ORF of 1005 base  pairs. Consistent with the results of the two-dimensional gel electrophoresis (Fig. 1), the cDNA sequence predicts a 334amino acid protein with a molecular mass of 38950.7 Da and a pI of 8.37 (Fig. 3A). The predicted protein sequence also contains the sequences of all other sequenced endo-R-C peptides (Fig. 3A). The sequence of MAYP shares high homology (87% identity and 95% similarity) to the sequence of a human EST clone (Genbank TM accession number AA082133) that is its presumed human homolog. The sequence of amino acids 6 -288 of MAYP bears 47% identity and 68% similarity to human proline, serine, threonine phosphatase interacting protein (PST PIP) (415 aa, molecular mass ϭ 47593.7 Da, pI ϭ 5.4) and 20% identity and 38% similarity to Saccharomyces cerevisiae CDC15p, which belong to a recently described family of PTP substrates. MAYP contains an NH 2 -terminal Fes/CIP4 homology (FCH) domain (aa 10 -98), followed by a coiled-coil domain (aa 93-121), containing sequences similar to those found in actin-binding domains of actin binding proteins (16,17), and a conserved basic and acidic acid-rich region (aa 99 -160). How-ever, it lacks the COOH-terminal SH3 domain of both PST PIP and CDC15 and the PEST domain of CDC15 (Fig. 3B). MAYP contains two potential SH3 binding, proline-rich sequences (PXXP) in the COOH-terminal region together with potential tyrosine, serine, and threonine phosphorylation sites. To further characterize MAYP, a rabbit antiserum was raised to a peptide corresponding an 18-aa sequence in the COOH-terminal region (Fig. 3A).
Expression of MAYP cDNA-Human embryonic kidney fibroblast 293T cells were transiently transfected with an expression plasmid containing the 2.1-kb p37 clone. The transfected plasmid drove the expression of a 37-kDa protein that was specifically recognized by the anti-MAYP peptide antibody (data not shown).
Tissue Distribution, Cell Line Expression, and Subcellular Localization of MAYP-Analysis of the expression of MAYP was carried out by immunoblotting. Its expression in cells was restricted to macrophage cell lines (BAC1.2F5, J774.2) and primary macrophages (BMM), save for very low expression in myelo-monocytic leukemic cells (WEHI-3) (Fig. 4A). Consistent with this pattern, it was detected at very low levels in macrophage-containing tissues, including bone marrow, spleen, liver, kidney, intestine, and brain (Fig. 4B). In BAC1.2F5 cells, Ͼ95% of MAYP was detected in the cytosol. However, ϳ4% was found in the Nonidet P-40-soluble membrane fraction and a trace amount in the Nonidet P-40-insoluble membrane fraction that was increased to ϳ2% by CSF-1 stimulation (Fig. 4C). No MAYP was detected in the nuclear fraction. The amounts of MAYP in the cytosol and membrane fractions are apparently unchanged following CSF-1 stimulation, and despite its presence in the membrane fraction, no association of MAYP with the CSF-1R could be detected (data not shown). However, when cells are incubated with IAA in the absence or presence of CSF-1, the increased tyrosine phosphorylation of total MAYP (Fig. 1 of Ref. 9; data not shown) is associated with a 5-fold increase in the proportion of MAYP in the membrane fraction (Fig. 4C).
CSF-1-stimulated MAYP Tyrosine Phosphorylation and Association with F-actin-Anti-Tyr(P) immunoblotting of MAYP immunoprecipitates of the cytosolic fraction of unstimulated macrophages and macrophages stimulated with CSF-1 at 37°C for 1 min revealed that MAYP is only very slightly tyrosinephosphorylated in unstimulated cells. However, in response to CSF-1 stimulation, the tyrosine phosphorylation of MAYP was increased by 6-fold (Fig. 5, upper panel). As expected from the results of the immunoblots in Fig. 4C, the amount of immunoprecipitable cytosolic MAYP was unchanged by CSF-1 stimulation (Fig. 5, middle panel). Previous studies have shown that phalloidin precipitation of F-actin from the cytosolic anti-Tyr(P)-reactive fraction co-precipitated a 37-kDa tyrosinephosphorylated protein (9), that we have subsequently identified to be MAYP by immunoblotting with the antiserum used for the experiments shown in Fig. 5 (data not shown). Immunoprecipitation of cytosolic MAYP also co-precipitated actin (Fig. 5, lower panel). These results indicate that MAYP associates, directly or indirectly, with F-actin. DISCUSSION Previous phalloidin co-precipitation experiments indicated that pp37 was the most prominent F-actin-associated tyrosinephosphorylated protein in the cytosolic fraction of CSF-1-stimulated macrophages (9). The present study establishes the identity of pp37 as MAYP and indicates that it is related to a group of PTP substrates, one of which has been shown to play an important role in regulation of the actin cytoskeleton. From its pattern of expression in cell lines, MAYP appears to be selectively expressed in macrophages (i.e. BAC1.2F5, BMM, and J774.2 cells), rather than less mature mononuclear phagocyte progenitor cells (i.e. M1 and WEHI-3 cells) and not at all in some other cell types, including fibroblasts, erythroid progenitors, and mast cells. MAYP exhibits a low level of tyrosine phosphorylation in unstimulated macrophages that is increased 4 -6-fold by stimulation with CSF-1. Although the vast majority of MAYP resides in the cytosolic fraction, ϳ4% resides in the membrane fraction and a trace amount in the cytoskeletal fraction. Upon stimulation with CSF-1, the proportion of MAYP in the cytoskeletal fraction is rapidly increased. Treatment with IAA alone increases the proportion of tyrosine-phosphorylated MAYP by 27-fold and increases the proportion of membrane associated MAYP by 6-fold. It is therefore possible that stimulation with CSF-1 alone, which increases tyrosine phosphorylation by only ϳ5-fold, is associated with a movement of MAYP to the membrane that is below the level of detection and that membrane association may require tyrosine phosphorylation of MAYP. Interestingly, cytosolic MAYP is constitutively associated with actin ( Fig. 5; Ref. 9). These data suggest that MAYP may be involved in regulating some of the rapid, CSF-1-induced cytoskeletal changes that take place within minutes of macrophage stimulation with CSF-1 (11,18,19).
MAYP, PST PIP, and CDC15 share FCH domains, coiled-coil domains, and basic and acidic amino acid-rich regions. PST PIP was identified by a yeast two-hybrid screen with the PEST-type PTP, PTP hematopoietic stem cell fraction (PTP-HSCF) (20), which is expressed in hematopoietic stem/progenitor cells and fetal thymus, but not in more differentiated cells, including macrophages (21,22). PST PIP appears to be the ortholog of the actin-associated Schizosaccharomyces pombe protein, CDC15p, a phosphorylated protein implicated in the assembly of the actin ring in the cytokinetic furrow (20,23). Association of PST PIP with PTP-HSCF involves the proline-rich region of the phosphatase and the coiled-coil domains of PST PIP. In cotransfection experiments in COS cells, PST PIP was shown to be tyrosine-phosphorylated by v-Src and dephosphorylated by its associated PTP-HSCF. PST PIP is co-localized with F-actinrich regions (cortical actin cytoskeleton, actin stress fibers, lamellopodia) in interphase cells and with cortical actin and the cytokinetic furrow in cells undergoing cytokinesis (20). More recently, its role in the regulation of the actin cytoskeleton was further emphasized by the demonstration that the interaction between the PST PIP SH3 domain and proline-rich regions of Wiskott-Aldrich syndrome protein (WASP) results in a loss of actin bundling activity by the COOH terminus of WASP and that tyrosine phosphorylation in the polyproline binding pocket of the SH3 domain of PST PIP inhibits binding of PST PIP and WASP, releasing WASP and PST PIP for their independent functioning elsewhere in the cell (24).
Three features of MAYP suggest that its function and regulation in cells differ from the function and regulation of PST PIP. First, MAYP appears to be selectively expressed in macrophages and within the mononuclear phagocytic lineage has the inverse expression pattern of PTP-HSCF, which is expressed in more primitive cells and not in macrophages (21,22). For this reason, MAYP is unlikely to be regulated by PTP-HSCF. Second, MAYP differs from PST PIP/CDC15 in that it lacks the SH3 domain of PST PIP that appears to be critical for its modulatory effect on WASP. Third, the existence of a human EST sharing 95% sequence similarity with MAYP indicates that MAYP is not the mouse homolog of human PST PIP.
Despite these differences, MAYP shares several features in common with PST PIP that suggest that they could function in a similar manner. First, they share a region of sequence similarity that includes an FCH domain, a coiled-coil domain, and a region rich in basic and acidic amino acids. Second, the increased tyrosine phosphorylation of MAYP in the presence of IAA could be due to inhibition of a closely associated PTP, akin to the regulation of PST PIP by PTP-HSCF. Third, MAYP binds F-actin, and PST PIP is clearly intimately involved in regulating the actin cytoskeleton.
FIG. 5. CSF-1-induced tyrosine phosphorylation of MAYP and its co-immunoprecipitation with actin. Cytosolic fractions from BAC1.2F5 cells that were either unstimulated (Ϫ) or stimulated (ϩ) with CSF-1 in the absence of IAA for 1 min at 37°C. Equal amounts of protein were subjected to immunoprecipitation with equivalent concentrations of F(abЈ) 2 fragments of either anti-p37 antibody or control IgG directly coupled to Affi-Gel beads. Immunoprecipitates were subjected to gradient SDS-PAGE and immunoblotting with antibodies to Tyr(P), MAYP, and actin. WB, Western blot.
The possibility that MAYP is involved in regulation of the actin cytoskeleton is appealing because of the presence of both the FCH domain and regions sharing sequence similarity with known actin-binding domains and because of the rapid reorganization of actin observed in response to CSF-1 (18,19). The FCH domain has been found to occur at the extreme NH 2 terminus of the nonreceptor tyrosine kinase FER, the Fujinami Sarcoma virus Fes/Fps family of proto-oncogene products, the RhoGAP protein p115, mouse proteins h74, and the growth arrest-specific gene product as well as two gene products from Caenorhabditis elegans (FO9E10.9 and F45E1.7) and one from S. cerevisiae. Several of these proteins have potential roles in organizing Rho proteins and the actin cytoskeleton. It has been suggested that the FCH domains may bind functionally related target molecules (25). The coiled coil domain of MAYP is highly conserved with the central coiled coil domain of PST PIP (67% identity). These domains are also rich in lysine and arginine residues and resemble sequences present in several actin-binding proteins (16,17), including paramyosin, myosin heavy chain, and troponin. While PST PIP has not been reported to bind actin, its function is intimately involved in the regulation of F-actin. Thus the association of MAYP with F-actin and its close relationship to PST PIP are intriguing, particularly in view of the rapid reorganization of the cytoskeleton observed in response to CSF-1, that follows MAYP tyrosine phosphorylation. Obviously, it will be important to identify the PTP(s) and kinase(s) that regulate MAYP tyrosine phosphorylation, the mechanism by which MAYP associates with actin and its functional role in the CSF-1 response of macrophages.