Plasma Membrane Phospholipid Scramblase 1 Promotes EGF-dependent Activation of c-Src through the Epidermal Growth Factor Receptor*

Phospholipid scramblase (PLSCR1) is a multiply palmitoylated, calcium-binding endofacial membrane protein proposed to mediate transbilayer movement of plasma membrane phospholipids. PLSCR1 is a component of membrane lipid rafts and has been shown to both physically and functionally interact with activated epidermal growth factor (EGF) receptors and other raft-associated cell surface receptors. Cell stimulation by EGF results in Tyr phosphorylation of PLSCR1, its association with both Shc and EGF receptors, and rapid cycling of PLSCR1 between plasma membrane and endosomal compartments. We now report evidence that upon EGF stimulation, PLSCR1 is phosphorylated by c-Src, within the tandem repeat sequence 68VYNQPVYNQP77. The in vivo interaction between PLSCR1 and Shc requires the Src-mediated phosphorylation on tyrosines 69 and 74. In in vitro pull down studies, phosphorylated PLSCR1 was found to bind directly to Shc through the phosphotyrosine binding domain. Consistent with the potential role of PLSCR1 in growth factor signaling pathways, granulocyte precursors derived from mice deficient in PLSCR1 show impaired proliferation and maturation under cytokine stimulation. Using PLSCR1–/– embryonic fibroblasts and kidney epithelial cells, we now demonstrate that deletion of PLSCR1 from the plasma membrane reduces the activation of c-Src by EGF, implying that PLSCR1 normally facilitates receptor-dependent activation of this kinase. We propose that PLSCR1, through its interaction with Shc, promotes Src kinase activation through the EGF receptor.

Phospholipid scramblase (PLSCR1) is a multiply palmitoylated, calcium-binding endofacial membrane protein proposed to mediate transbilayer movement of plasma membrane phospholipids. PLSCR1 is a component of membrane lipid rafts and has been shown to both physically and functionally interact with activated epidermal growth factor (EGF) receptors and other raft-associated cell surface receptors. Cell stimulation by EGF results in Tyr phosphorylation of PLSCR1, its association with both Shc and EGF receptors, and rapid cycling of PLSCR1 between plasma membrane and endosomal compartments. We now report evidence that upon EGF stimulation, PLSCR1 is phosphorylated by c-Src, within the tandem repeat sequence 68 VYNQPVYNQP 77 . The in vivo interaction between PLSCR1 and Shc requires the Srcmediated phosphorylation on tyrosines 69 and 74. In in vitro pull down studies, phosphorylated PLSCR1 was found to bind directly to Shc through the phosphotyrosine binding domain. Consistent with the potential role of PLSCR1 in growth factor signaling pathways, granulocyte precursors derived from mice deficient in PLSCR1 show impaired proliferation and maturation under cytokine stimulation. Using PLSCR1؊/؊ embryonic fibroblasts and kidney epithelial cells, we now demonstrate that deletion of PLSCR1 from the plasma membrane reduces the activation of c-Src by EGF, implying that PLSCR1 normally facilitates receptor-dependent activation of this kinase. We propose that PLSCR1, through its interaction with Shc, promotes Src kinase activation through the EGF receptor.
Phospholipid scramblase 1 (PLSCR1) 1 is an endofacial plasma membrane protein that has been proposed to play a role in the redistribution of plasma membrane phospholipids leading to cell surface exposure of phosphatidylserine following cell activation, injury, or apoptosis (1)(2)(3). There are four distinct human PLSCR genes (hPLSCR1-4), corresponding orthologous genes in mouse (mPLSCR1-4), and putative orthologues in Drosophila, Caenorhabdites elegans, and Saccharomyces cerevisiae (4).
The amino acid sequence of human PLSCR1 reveals a type-2 membrane protein containing 318 residues with a predicted transmembrane helix near the C terminus (2)(3)(4). The cytoplasmic domain includes an EF hand-like calcium-binding domain and a predicted protein kinase C phosphorylation site (3,4). PLSCR1 is multiply Cys-palmitoylated (5,6) and has been shown to partition into lipid rafts, cholesterol and sphingolipidrich membrane microdomains implicated in regulation and endocytic trafficking of receptor-signaling complexes (7). The N-terminal cytoplasmic domain of PLSCR1 contains multiple proline-rich motifs resembling both Src homology 3 (SH3) domain-binding sites and WW domain-binding motifs (4). PLSCR1 has been shown to bind c-Abl through its SH3 domain and to be a substrate of activated c-Abl tyrosine kinase (8).
Mice with targeted disruption of the PLSCR1 locus exhibit delayed fetal production of mature blood granulocytes and defective granulocytosis in response to stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) but not to interleukin 3 or granulocyte macrophage colony-stimulating factor (9). Consistent with these in vivo data, myeloid precursor cells from PLSCR1Ϫ/Ϫ mice in colony-forming assays display impaired proliferation and maturation when cultured with either SCF or G-CSF (9). Stimulation of myeloid precursor cells by either SCF or G-CSF was also found to induce marked increases in cellular expression of PLSCR1, suggesting that it is transcriptionally regulated through these growth factors (9). PLSCR1 is also one of the most highly interferon-induced genes and was shown to be transcriptionally regulated by signal transducer and activator of transcription 1 (STAT-1) and by an interferon-stimulated response element in its first exon (10).
In cells expressing receptors for epidermal growth factor (EGF), stimulation with EGF leads to an association of PLSCR1 with both activated EGF receptors (EGF-R) and with the adaptor protein Shc, and results in increased Tyr phosphorylation of PLSCR1 (7). EGF also induces the internalization of PLSCR1 from the plasma membrane in conjunction with EGF-R. Whereas EGF-R is subsequently ubiquitinated and degraded, PLSCR1 was found to recycle from its endosomal compartment to the cell surface (7).
To gain insight into the functional significance of the observed interaction of PLSCR1 with EGF-R and related growth factor receptors, we have examined EGF signaling in cells derived from PLSCR1Ϫ/Ϫ mice (9). Our data indicate that an activated Src kinase is responsible for the Tyr phosphorylation of PLSCR1 observed in EGF-treated cells and that this phos-phorylation is required for the interaction of PLSCR1 with Shc upon EGF stimulation. Furthermore, our data suggest that deletion of PLSCR1 from the plasma membrane results in marked reduction in EGF-initiated activation of c-Src kinase.

EXPERIMENTAL PROCEDURES
Antibodies and Reagents-The murine anti-PLSCR1 monoclonal antibody (mAb) 4D2 (specific for human PLSCR1) has been previously described (8) and recognizes a single protein of 37 kDa in cell lysates. The mAb 1A8, recognizing mouse PLSCR1, has been developed in our laboratory and does not cross react with other PLSCR proteins (PLSCR2-4) (data not shown). Rabbit anti-EGF-R (1005) polyclonal IgG and HRP-conjugated anti-Tyr(P) mAb PY99 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cdc2 peptide, purified recombinant human c-Src enzyme, and rabbit anti-Shc polyclonal IgG against p46, p52, and p66 were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-c-Src mAb 327 was from Oncogene (San Diego, CA), and anti-c-Src mAb 2-17 antibody was a kind gift of Dr. Sally Parsons (University of Virginia, Charlottesville, VA). Appropriate HRPconjugated secondary antibodies were obtained from Jackson Immuno-Research Laboratories (West Grove, PA). Anti-maltose-binding protein (MBP) rabbit polyclonal antibody was obtained from New England Biolabs (Beverly, MA).
Cell culture media and reagents were purchased from Invitrogen, and complete EDTA-free Protease Inhibitor Mixture Tablets (containing inhibitors for serine and cysteine proteases) was from Roche Applied Science. AG1478, an EGF-R tyrosine kinase inhibitor, and PP2, a Src family tyrosine kinase inhibitor, were obtained from Calbiochem (San Diego, CA), and EGF was from BD Biosciences (San Diego, CA). The Abl inhibitor, STI571, was the gift of Dr. Elizabeth Buchdunger (Novartis Pharma AG, Basel, Switzerland). The PLSCR family members, hPLSCR1, 2, 3, and 4, were produced as fusion proteins with MBP as described previously (2).
Preparation of Cell Lysates and Immunoprecipitation-Cells were starved in serum-free medium for 3 to 14 h prior to stimulation with EGF (100 ng/ml) for the indicated times. In some experiments, cells were preincubated with PP2, at the indicated concentrations, for 1 h prior to EGF stimulation. After treatment of the sample with growth factor, the medium was aspirated and the cells were rapidly chilled by washing with ice-cold phosphate-buffered saline. Cells were lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM dithiothreitol, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 5 mM EDTA, and protease inhibitor mixture) on ice for 30 min. Cell lysates were centrifuged at 10,000 ϫ g for 10 min at 4°C, and the supernatants were transferred to fresh tubes for further use.
For immunoprecipitation, cell lysates containing 100 -500 g of protein were incubated with 2 g of anti-c-Src mAb 327 or mAb 2-17, or 5 g of anti-hPLSCR1 mAb 4D2, 2 g of anti-Shc polyclonal IgG, or 2 g of anti-EGF-R polyclonal IgG for 2 h on ice. Protein G-Sepharose 4 Fast Flow beads were added to precipitate the immune complex by rotating at 4°C for 1 h. Beads were pelleted at 1000 ϫ g for 2 min and washed extensively, and immunoprecipitates were subjected to Western blotting or in vitro kinase assays.
In Vitro Tyrosine Kinase Assay-EGF-R or c-Src immunoprecipitated from MEF, KEC, or A431 cell lysates, or purified recombinant c-Src as indicated in the figure legends, were used for in vitro tyrosine phosphorylation of PLSCR1. In a total volume of 30 l of reaction mixture, 4 g of MBP-PLSCR1 fusion protein or 1-4 g of cdc2 peptide was used as substrate in kinase assay buffer containing 10 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , 1.2 mM MnCl 2 , and 2 mM sodium orthovanadate. The reactions were initiated by the addition of 2 l of 1 mM cold ATP or 2 l of 8 M ATP containing 10 Ci of [␥-32 P]ATP. After incubation for 15 min at room temperature (for A431 cells), or 1 h at 37°C (for MEFs, KECs, and purified recombinant c-Src), reactions were terminated by adding SDS-PAGE sample buffer and boiling at 100°C for 5 min. Proteins were resolved by SDS-PAGE, and phosphorylation was detected by Western blotting with anti-Tyr(P) mAb PY99-HRP or autoradiography.
In Vitro Binding of PLSCR1 to GST Fusion Proteins-The GST fusion proteins of full-length p52 Shc were kindly provided by Dr. Ralph Bradshaw (University of California at Irvine, Irvine, CA) and those of the phosphotyrosine binding (PTB), collagen homology (CH), and SH2 domains of Shc by Dr. Kodi Ravichandran (Beirne Carter Center for Immunology Research, University of Virginia). The GST fusion proteins were expressed by induction overnight at 25°C with 0.1 mM isopropyl-1-thio-␤-D-galactopyranoside in BL21 DE3 (protease deficient) Escherichia coli and purified using glutathione-Sepharose 4B according to the manufacturer's instructions.
MBP-PLSCR1 was purified as previously reported (8) and digested with factor Xa to release MBP. 5 g of PLSCR1 was then phosphorylated in vitro by incubation with 5 units of purified c-Src for 9 h at room temperature in kinase reaction buffer containing 10 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , 1.2 mM MnCl 2 , 2 mM sodium orthovanadate, and 2 mM ATP. For a typical in vitro binding assay, 5 g of GST fusion protein of full-length p52 Shc or Shc domains immobilized on glutathione-Sepharose 4B beads were incubated with 5 g phosphorylated PLSCR1 in 100 l binding buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2% Nonidet P-40, 0.1% bovine serum albumin, 1 mM dithiothreitol, protease inhibitor mixture, 10 mM sodium fluoride, and 2 mM sodium orthovanadate) overnight at 4°C with gentle rotation. The beads were sedimented by centrifugation and washed extensively with binding buffer followed by washing in phosphate-buffered saline. Bound proteins were eluted with 30 l of SDS-PAGE sample buffer containing 0.1 mM dithiothreitol, resolved on a 10% Tris glycine gel, and analyzed by Western blotting.

RESULTS
Phosphorylation of PLSCR1 in Response to EGF Is Mediated by a Src kinase-We had previously observed that cellular treatment with EGF results in a transient increase in Tyr phosphorylation of PLSCR1 (7). An increase in PLSCR1 phosphorylation was also observed for platelet-derived growth factor and SCF, suggesting that PLSCR1 might be a substrate of a tyrosine kinase(s) that is activated through multiple growth factor receptors (14) (data not shown). This indicated that PLSCR1 was a substrate of either the growth factor receptor tyrosine kinases themselves or of other tyrosine kinase(s) activated through EGF-R, platelet-derived growth factor receptor, or c-kit, respectively, including potentially the Src family of tyrosine kinases that are known to function prominently in the growth factor receptor activation pathways (15). As shown in Fig. 1, Tyr phosphorylation of PLSCR1 upon cell treatment with EGF was found to be sensitive to inhibition by the Src kinase inhibitor PP2 at concentrations of inhibitor that had only small effects on the autophosphorylation of EGF-R, suggesting that a Src kinase mediates phosphorylation of PLSCR1 under these conditions. Consistent with these data, when recombinant PLSCR1 was used as substrate for in vitro kinase assays of either immunoprecipitated EGF-R ( Fig. 2A) or c-Src (Fig. 2B), only c-Src was found to mediate the phosphorylation of PLSCR1. Because Src kinases are known to phosphorylate and activate c-Abl (16), a tyrosine kinase that was previously shown to phosphorylate PLSCR1, we also considered the possibility that contaminating c-Abl in the c-Src immunoprecipitates might account for the observed phosphorylation of PLSCR1. Whereas phosphorylation of PLSCR1 was prevented in the presence of the Src family kinase inhibitor, PP2, the Abl kinase inhibitor, STI571, failed to inhibit phosphorylation by immunoprecipitated c-Src, which further implicates c-Src as the kinase responsible for phosphorylating PLSCR1 under these conditions (Fig. 2B). Additional supporting evidence for c-Src as the kinase involved was obtained using purified recombinant human c-Src, expressed and purified from Sf9 insect cells, which was also found to phosphorylate PLSCR1 (Fig. 3A) and was sensitive to the Src kinase family inhibitor, PP2 (Fig.  3A). Among the known human PLSCR family members, only PLSCR1 was observed to be phosphorylated by purified c-Src (Fig. 3B).
Identification of the Sites of c-Src Phosphorylation in PLSCR1-The sites of phosphorylation of PLSCR1 by c-Src were next mapped using Tyr mutants of the PLSCR1 polypeptide. We found that mutation at both Tyr 69 and Tyr 74 was required to eliminate PLSCR1 phosphorylation by both Src immunoprecipitated from cell lysates (results not shown) and purified recombinant c-Src (Fig. 4A), or in EGF-stimulated cells transfected with wild type (WT) or mutant PLSCR1 (Fig. 4B). These Tyr residues are within the same tandem repeat sequence 68 VYNQPVYNQP 77 that we have previously identified as the sites required for phosphorylation of PLSCR1 mediated by c-Abl. Mutation at either Tyr 69 or Tyr 74 markedly reduced but did not abolish the phosphorylation by c-Src, whereas phosphorylation was eliminated when both residues were replaced with Phe ( Fig. 4 and data not shown), implying the potential for phosphorylation at both sites.
The EGF-mediated Interaction between PLSCR1 and Shc Requires Phosphorylation of Tyr 69 and Tyr 74 in PLSCR1-We have previously observed increased association of PLSCR1 with both the adaptor protein Shc and EGF-R in response to cell treatment with EGF (7). Therefore, we investigated whether the increased binding of PLSCR1 to Shc adaptor protein is dependent on the c-Src-mediated phosphorylation of PLSCR1. As shown in Fig. 5, in KECs expressing wild type PLSCR1, stimulation with EGF resulted in increased association of PLSCR1 with Shc. In contrast, little or no association with Shc was observed for mutant Y69F/Y74F PLSCR1 upon cell treatment with EGF. These results clearly indicate that the EGF-induced association between PLSCR1 and Shc requires the c-Src phosphorylation site(s) Tyr 69 and Tyr 74 of PLSCR1.
In Vitro Studies Implicate the PTB Domain of Shc in Its Interaction with PLSCR1-We next set out to identify the domain(s) within Shc responsible for its interaction with Tyr phosphorylated PLSCR1. All Shc isoforms contain a PTB and a Src homology 2 (SH2) domain, motifs known to mediate protein-protein interactions by binding to sequences containing phosphotyrosines (17)(18)(19)(20). Full-length p52 Shc, and the PTB, CH, and SH2 domains of Shc were expressed as GST fusion proteins and used to interact with PLSCR1 that had been phosphorylated by c-Src. Of the various domains of Shc, only full-length p52 Shc and the PTB domain of Shc were observed to bind phosphorylated PLSCR1 (Fig. 6). Consistent with this, Tyr(P) PLSCR1 was depleted in the supernatants after pull down with full-length Shc and PTB, compared with the GST control and the other Shc domains tested. These results suggest that interaction of phosphorylated PLSCR1 with Shc is mediated predominantly by the PTB domain. In the absence of phosphorylation, some interaction of PLSCR1 with full-length Shc and its PTB domain was also observed (see "Discussion"). Attenuated Activation of c-Src Kinase in Cells Deficient in PLSCR1-Cell stimulation with EGF leads to Tyr phosphorylation of PLSCR1, its increased association with Shc and EGF-R, as well as prominent endosomal recycling of PLSCR1 coincident with endocytosis of the receptor (7). This suggests that PLSCR1 might play some functional role in the growth factor receptor activation or signaling pathways. Consistent with this possibility, hematopoietic cells derived from PLSCR1Ϫ/Ϫ mice show defects in proliferation and maturation in response to either G-CSF or SCF, growth factors central to normal myeloid development (9). Because PLSCR1 was found to be phosphorylated by c-Src, one of a family of enzymes that are central to intracellular signaling through multiple growth factor pathways (15), we considered the possibility that the activation of Src kinase through EGF-R might itself be affected by deletion of PLSCR1. As illustrated by Fig. 7, the EGF-dependent phosphorylation and activation of c-Src kinase was markedly attenuated in cells with a targeted deletion of the PLSCR1 gene locus, when compared with identically matched cells expressing PLSCR1, implying that PLSCR1 normally functions in the activation of c-Src by stimulated EGF-R. The apparent difference between PLSCR1-expressing versus PLSCR1-deficient cells in the growth factor-dependent activation of c-Src was consistently observed in multiple cell lines derived from independent transformants of PLSCR1Ϫ/Ϫ MEFs and KECs that were infected with either vector or PLSCR1. DISCUSSION We have previously shown that upon EGF stimulation, PLSCR1 is Tyr phosphorylated and associates with both the Shc adaptor protein and the EGF receptor (7). The present study establishes that 1) the EGF-induced phosphorylation of PLSCR1 involves a Src kinase, 2) the sites of Src phosphorylation in PLSCR1 involve Tyr 69 and Tyr 74 , 3) the interaction between PLSCR1 and Shc requires phosphorylation of Tyr 69 and Tyr 74 on PLSCR1, 4) Shc interacts with phosphorylated PLSCR1 through its PTB domain, and 5) deletion of PLSCR1 substantially attenuates the growth factor-dependent activation of c-Src, suggesting that PLSCR1 normally facilitates c-Src activation through EGF-R and potentially other related growth factor receptors.
Because many of Src's substrates bind through either the SH2 or SH3 domains (15,16,(21)(22)(23), we postulated that PLSCR1 might interact with Src in a similar manner, as it contains several PXXP motifs that could potentially serve as binding sites for the SH3 domain of Src. Nevertheless, we were unable to demonstrate association of PLSCR1 with Src through co-immunoprecipitation studies (data not shown), suggesting either a low affinity interaction that is disrupted under the conditions of immunoprecipitation, or that another adaptor molecule may be required as a bridge between PLSCR1 and Src. Upon EGF stimulation, increased phosphorylation of PLSCR1 and its association with both EGF-R and the adaptor protein Shc was observed (7). Our present data now suggest that Tyr 69 and Tyr 74 of PLSCR1, which are required for phosphorylation by Src, are also required for PLSCR1's interaction with Shc (Fig. 5), and we demonstrate that phosphorylated PLSCR1 interacts with Shc through Shc's PTB domain (Fig. 6). Because tyrosine phosphorylation is a reversible, dynamic process controlled by the activities of protein kinases and the competing actions of protein tyrosine phosphatases, many SH2/ PTB domain-phosphotyrosine-mediated interactions are shortlived. This is also the case for EGF-induced phosphorylation of PLSCR1 (Fig. 1), and its consequent recruitment into the EGF-R signaling complex mediated through its transient association with Shc (Fig. 6). Although we also noted some binding of unphosphorylated PLSCR1 to the PTB domain of Shc in in vitro pull down assays using recombinantly expressed proteins, such interaction does not appear to occur to a significant extent within the cell (Fig. 5). Because Shc isoforms are predominantly localized to the cytoplasm in quiescent cells and translocate to the plasma membrane only upon EGF stimulation (24,25), increased association of plasma membrane-bound Shc with membrane PLSCR1 after EGF stimulation might be expected simply due to their increased proximity. Their interaction appears to be not only dependent on co-localization at the plasma membrane, but also on phosphorylation of PLSCR1 at Tyr 69 and Tyr 74 , because intracellular association of mutant Y69F/ Y74F PLSCR1 with Shc was not observed (Fig. 5). One possibility is that upon EGF stimulation, phosphorylated PLSCR1 initially recruits Shc to the plasma membrane through this interaction with the Shc PTB domain, and then promotes transfer of Shc to its phosphotyrosine-binding site in EGF-R (Tyr 1173 /Tyr 1148 in EGF-R) (26). Alternatively, phosphorylated PLSCR1 might serve to displace Shc from the activated receptor through its competition for the Shc PTB domain, and thereby promote throughput of new substrate that is delivered to the receptor kinase.
It is of interest that in addition to attenuated activation of c-Src upon EGF stimulation, cells deficient in PLSCR1 consistently exhibited reduced basal Src kinase activity (Fig. 7). Presumably, this either reflects incomplete receptor inactivation through serum starvation, or some direct effect of PLSCR1 on the constitutive activity of c-Src. In this context, plasma membrane-recruited Shc has recently been implicated in the direct activation of c-Src (27). One possibility is that the small pool of PLSCR1 that is constitutively phosphorylated by c-Abl on Tyr 69 and Tyr 74 (8) may serve to recruit Shc to the plasma membrane in quiescent cells, thereby facilitating interactions between Shc and membrane-localized c-Src, increasing basal Src kinase activity. Upon EGF stimulation, further activation of c-Src is thought to be promoted through interaction of phosphorylated EGF-R with the Src SH2 domain (14). This activated c-Src is then capable of phosphorylating EGF-R (15), Shc (28), and PLSCR1 (Figs. 1-4). After EGF stimulation, the increased pool size of phosphorylated PLSCR1 may then recruit more Shc to the plasma membrane, leading to further activation of Src by Shc. Thus, a reduced amount of plasma membrane-localized Shc might account for the attenuated activity of c-Src observed in the PLSCR1 deficient cells.
As previously noted, cells deficient in PLSCR1 have been FIG. 7. EGF-induced c-Src kinase activity is reduced in PLSCR1؊/؊ cells. PLSCR1Ϫ/Ϫ MEFs (A) and KECs (B) stably expressing PLSCR1, or vector control were stimulated with 100 ng/ml EGF for times shown. Cell lysates were prepared and c-Src was immunoprecipitated. Src kinase activity in the immunoprecipitates was measured using [␥-32 P]ATP and the Src-specific substrate cdc2 peptide. Cdc2 peptide was resolved on 16% tricine gels and detected by autoradiography. Data shown are representative of 4 experiments performed.
FIG. 6. PLSCR1 binds to Shc through the PTB domain in vitro. PLSCR1 was phosphorylated in vitro using purified recombinant c-Src (see "Experimental Procedures"). After incubation of either phosphorylated or unphosphorylated PLSCR1 with Shc domains immobilized on glutathione 4B-Sepharose beads, an aliquot of the supernatant was retained and the bound proteins were eluted. These samples were subjected to Western blot analysis. Phosphorylated PLSCR1 was detected with anti-Tyr(P) antibody, and total PLSCR1 with anti-PLSCR1 mAb 4D2. Amounts of GST fusion proteins immobilized on Sepharose 4B beads were detected by Ponceau stain. Stars denote GST-fusion proteins of correct molecular weight (p52 and CH domain contained proteolytic breakdown products) (29). Data shown are representative of 2 experiments performed. observed to exhibit defects in proliferative and maturational responses to selective growth factors, suggesting that this protein might play some role in signaling through growth factor receptors (9). Our present data suggest that at least one explanation for the phenotype that has been observed in PLSCR1Ϫ/Ϫ cells relates to a specific role of PLSCR1 in promoting the kinase activity of cellular c-Src.