(Received for publication, July 18, 1995)
From the
Phosphorylation and dephosphorylation of Tyr-530 in human c-Src
(Tyr-527 in avian c-Src) is critical in regulating c-Src kinase
activity. So far, it has not been possible to distinguish the active
and inactive forms in vivo. We now report a new monoclonal
antibody that selectively recognizes the active form of c-Src. This
antibody, termed clone 28, recognized a region adjacent to Tyr-530
(QYQP
) in the C-terminal regulatory domain
of c-Src, and its binding was hindered by phosphorylation of this
tyrosine as determined by peptide competition assay. Combined
immunoprecipitation/Western blotting revealed that clone 28 reacted
with a 60-kDa protein that was precipitated by mAb 327, a well known
monoclonal antibody against v-Src and c-Src. Cyanogen bromide cleavage
and two-dimensional tryptic maps confirmed that clone 28 was specific
for the active form (Tyr-530 not phosphorylated), whereas mAb 327
recognized the inactive form (Tyr-530 phosphorylated) as well as the
active form. Clone 28 selectively immunoprecipitated the active form
and augmented its kinase activity. Preabsorption experiments revealed
that clone 28 could not completely immunoprecipitate the mAb 327
binding 60-kDa protein in either an in vitro or an in vivo phosphorylation system. These observations, taken together,
strongly suggest the existence of multiple forms of c-Src as proposed
by Cooper and Howell(1993) (Cooper, J. A., and Howell, B.(1993) Cell 73, 1051-1054).
Using clone 28, we demonstrated a distinct localization of the active form of c-Src within cultured normal fibrobast cells. In liver tissue sections, we also examined the distribution of the active form in embryonic mice. Megakaryocytes were strongly stained, in contrast to completely negative immunoreactivity in hepatocytes, reticulocytes, and granulocytes. This result provides the first direct evidence that c-Src is highly activated in platelets.
Src family tyrosine kinases are widely distributed nonreceptor
tyrosine kinases that have a role in many different signal transduction
pathways downstream from various types of receptor in the cell
membrane; however, their exact roles in signal transduction remain
unknown. Src family tyrosine kinases (for reviews, see Hunter(1987),
Cantley et al.(1991), and Bolen et al.(1992)) are
known both from retroviruses that have the ability to transform cells
(Src, Yes, Fgr, and Lck) and from mammalian genomes by the use of DNA
probes for src/yes/fgr (Fyn, Lyn, Hck, Blk, and Yrk). A
comparison of the corresponding cDNA sequences revealed that each
member of this family has a very different sequence in the N-terminal
region (50-80 amino acids from the N terminus). This region is
thought to participate in specific cellular function through binding to
different types of signal transducer (Resh, 1989; Shaw et al.,
1990; Timson-Gauen et al., 1992), although the mechanism of
regulation remains unknown. By contrast, the rest of the sequence is
highly conserved, consisting sequentially of an Src homology (SH) ()3 domain, an SH2 domain, a kinase domain, and a C-terminal
regulatory domain (for reviews, see Margolis(1992), Pawson and
Gish(1992), Cooper and Howell(1993), and Erpel and Courtneidge(1995)).
Two tyrosine residues, Tyr-419 and Tyr-530 in human c-Src (corresponding to Tyr-416 and Tyr-527, respectively, in avian c-Src), can be phosphorylated, and their phosphorylation state influences the tyrosine kinase activity. Whereas the phosphorylation of Tyr-419 leads to a dramatic increase in kinase activity (Piwnica-Worms et al., 1987), the phosphorylation of Tyr-530 negatively regulates the kinase activity (Cooper et al., 1986). The csk gene product is responsible for the phosphorylation of Tyr-530 (Okada and Nakagawa, 1989; Okada et al., 1991). The mechanism of regulation involving these tyrosine residues is thought to be similar for other members of the Src family (Okada et al., 1991). On the basis of these observations, a model that involves intramolecular binding between Tyr-530 and the SH2 region was proposed (Roussel et al., 1991; Superti-Furga et al., 1993; Sieh et al., 1993). In this widely accepted model, subsequent signal transduction through direct binding to other proteins or by tyrosine phosphorylation is assumed to be prevented by this intramolecular binding. Consequently, the C-terminal region is important not only in the regulation of tyrosine kinase activity, but also in the association with SH2-containing proteins.
In the present study, we have generated a monoclonal antibody (mAb) specific for the C-terminal regulatory domain of c-Src. This mAb selectively recognized the active form of c-Src as demonstrated by in vitro and in vivo phosphorylation. Several different types of experiment described here illustrated the usefulness of this mAb. The antibody had a different range of reactivity in comparison with mAb 327, which has been widely used for the detection of c-Src and v-Src. In particular, clone 28 was shown to have high sensitivity to the active form of c-Src in normal cells and tissues.
Figure 1: Reactivity of clone 28 and mAb 327 with human platelet lysates. A, Western blotting with control mouse IgG (lane 1), clone 28 (lane 2), and mAb 327 (lane 3) in human platelets. B, combined immunoprecipitation/Western blotting in human platelets. Platelet lysates were immunoprecipitated with control mouse IgG (lane 1), clone 28 (lanes 2 and 3), and immune complexes were subjected to electrophoresis for Western blotting by mAb 327 (lanes 1 and 2) or clone 28 (lane 3).
Figure 2:
Detection of phosphorylated proteins from
human platelets. A, in vivo phosphorylation assay.
Platelets were metabolically labeled with
[P]PO
, then
immunoprecipitated with control mouse IgG (lane 1, 10 µg),
clone 28 (lane 2, 10 µg; lane 3, 1 µg; lane 4, 0.1 µg), or mAb 327 (lane 5, 10 µg; lane 6, 1 µg; lane 7, 0.1 µg). B, in vitro phosphorylation assay. In vitro kinase
activity was assessed in human platelet lysates immunoprecipitated by
control mouse IgG (lane 1, 10 µg), clone 28 (lane
2, 10 µg; lane 3, 1 µg; lane 4, 0.1
µg), or mAb 327 (lane 5, 10 µg; lane 6, 1
µg; lane 7, 0.1 µg).
Figure 3: Competition for antibody binding in in vitro phosphorylation systems. We synthesized a series of short peptides as illustrated in Table 1and determined their ability to inhibit immunoprecipitation by clone 28 in the in vitro kinase assay (A). Immunoprecipitation was performed in the presence of 0.1 mg/ml LEDYFTST (519-526, lane 1), EPQYQPG (527-533, lane 2), QYQPGENL (529-536, lane 3), QF*QPGENL (529-536, lane 4), or QYQPGD*Q*T* (529-536, lane 5) or in the absence of synthetic peptides (lane 6). We also compared the inhibition of immunoprecipitation caused by the non-phosphorylated peptide QYQPGENL (B, lanes 2-4) and the corresponding phosphotyrosine-containing peptide (B, lanes 5-7). The concentration of each peptide was 1 µg/ml (lanes 2 and 5), 10 µg/ml (lanes 3 and 6), or 100 µg/ml (lanes 4 and 7). The control level was determined in the absence of any peptide (lane 1).
Figure 4: Phosphopeptide mapping. Excised 60-kDa bands from lanes 2 (clone 28 precipitate) and 5 (mAb 327 precipitate) in Fig. 2A were subjected to V8 protease partial digestion (A) or cyanogen bromide cleavage (B). Lanes 1 correspond to clone 28 and lanes 2 to mAb 327. Excised V1 and V2 bands from each lane in A were further analyzed by two-dimensional tryptic peptide mapping (C).
We also
performed preabsorption experiments in in vitro and in
vivo systems. Lysates of human platelets metabolically labeled
with [P]PO
were
pretreated with normal mouse IgG or clone 28, then subjected to clone
28 and mAb 327 precipitation (Fig. 5A). Lanes 1 and 2 show the basal level of clone 28 and mAb 327
binding activity toward the phosphorylated 60-kDa form when normal
mouse IgG was used in preabsorption step. Even when excess clone 28 was
used in the preabsorption step, substantial phosphorylated 60-kDa bands
remained that were recognized by mAb 327 (lane 3). This result
suggested that substantial amounts of c-Src were phosphorylated at
Tyr-530 in human platelets. (If a significant amount of the observed
phosphorylation were due to Ser or Thr, we would have expected a
reduction in the intensity of the 60-kDa band (see Fig. 4A).) In addition, we pretreated cold platelet
lysates with normal mouse IgG or clone 28 and then subjected them to
clone 28 and mAb 327 precipitation (Fig. 5B). Under
conditions when both clone 28 and mAb 327 showed high
autophosphorylation activity (lanes 1 and 2),
substantial kinase activity was observed in the mAb 327 precipitate
even when excess clone 28 was used in the preabsorption step (lane
3).
Figure 5: Preabsorption of human platelet lysates. Human platelet lysates were pretreated with control mouse IgG (lanes 1 and lanes 2) or clone 28 (lanes 3). After preabsorption, lysates were further incubated with clone 28 (lanes 1) or mAb 327 (lanes 2 and lanes 3). In vivo (A) or in vitro (B) phosphorylation assays were performed as in Fig. 2.
Figure 6: The distribution of c-Src within normal rat fibroblast 3Y1 cells. Comparison between clone 28 and mAb 327 in an immunocytochemical analysis. The 3Y1 cells were stained with clone 28 (A) or mAb 327 (B). Bar, 15 µm.
In order to test the
utility of the clone 28 directly in an in vivo system, we
examined formalin-fixed, paraffin-embedded C3H-mouse fetal liver (16
days) by an immunocytochemical method. Fig. 7clearly shows the
presence of the active form of c-Src in megakaryocytes, but completely
negative immunoreactivity in hepatocytes, reticulocytes, and
granulocytes in the fetal liver. We also detected high level of the
active form in neuronal tissues. ()
Figure 7: Immunoreactive localization of the active form of c-Src in the liver of developing mouse embryo (16 days). Sections were stained with clone 28 (A) or normal mouse IgG (B, as a negative control). The active form of c-Src was found preferentially in inside the plasma membrane of megakaryocytes. Bar, 20 µm.
In order to clarify the activation mechanism of Src family tyrosine kinases, we set out to produce mAbs that could be used to distinguish the active and inactive forms of these tyrosine kinases in various systems. Activation is associated with dephosphorylation of the tyrosine residue nearest the C terminus (Courtneidge, 1985; Cooper and King, 1986), and subsequent signal transfer is presumed to involve a dissociation between this tyrosine-containing region and an SH2 region. We have generated a single mAb, termed clone 28, that could selectively recognize the active form of c-Src. We have characterized clone 28 and demonstrated its usefulness as follows: 1) clone 28 sensitively detected the active form of c-Src as judged by titration in an in vitro phosphorylation experiment (Fig. 2); 2) a competition study using phosphorylated and nonphosphorylated synthetic peptides confirmed the specificity of clone 28 for the active (Tyr-530 nonphosphorylated) form (Fig. 3); 3) the phosphorylation site of the clone 28 precipitate was clearly distinguishable from that of the mAb 327 precipitate (Fig. 4); 4) preabsorption experiments with clone 28 and mAb 327 in in vitro and in vivo phosphorylation systems suggested that there are at least two different conformations that retain kinase activity (Fig. 5); and 5) the distribution of the active form in rodent tissues and cultured cells could be directly observed by an immunocytochemical method ( Fig. 6and 7).
Cooper and Howell(1993) proposed a new activation mechanism for Src family tyrosine kinases involving allosteric activators or inhibitors. They presumed that phosphorylation of the tyrosine residues might be a consequence, and not a cause, of changes in activity. Site-directed mutagenesis studies (Hirai and Varmus, 1990; O'Brien et al., 1990; Seidel-Dugan et al., 1992) support this notion, because Src could be activated by introducing mutations in other domains such as SH2 or SH3. The detection of kinase activity both before and after preabsorption by clone 28 (Fig. 5) also showed good agreement with this previous work, since excess clone 28 could not completely immunoprecipitate the active form, even though this clone was specific for the active form. Erpel and Courtneidge(1995) also discussed yet another activation mechanism and proposed three possible pathways to the active form.
In platelets, V8 phosphopeptide mapping facilitated further analysis of the phosphorylation state of the clone 28 and the mAb 327 precipitates: the patterns for both precipitates were virtually identical (Fig. 4A). Cichowski et al.(1992) detected Src, Yes, and Fyn, but not Fgr, in thrombin-stimulated human platelets. They co-precipitated Yes and Fyn with an antibody against GTP-activating protein, but the member of the Src family responsible for platelet activation has not been identified. We also investigated several antibodies against each member of the c-Src family in order to identify which were active in the platelet system, but none of the antibodies demonstrated satisfactory sensitivity or specificity. However, clone 28 could give us valuable information on alterations in the kinase activity and cellular localization of Src family tyrosine kinases; such information could help us understand the redundancy and compensation in vivo among the members of the Src family that possess the QYQPG motif. Primary cells derived from knockout mice (e.g. src, fyn, and yes) might provide information about the role of each member of the Src family when tested with our new antibody.
Polyclonal antibodies against the C-terminal region of c-Src have previously been reported (Courtneidge and Smith, 1984; Cooper and King, 1986). One of these antibodies (Courtneidge and Smith, 1984) could be used both for the precipitation of the phosphorylated form of the kinase and for the autophosphorylation assay. However, there was no mention of cross-reactivity among members of the Src family. Another antibody (Cooper and King, 1986) stimulated the kinase activity upon antibody binding and could also be used for the precipitation of the phosphorylated form. For these polyclonal antibodies, it is possible that several epitopes coexist, some containing Tyr-530 and some not. The results obtained with these antibodies are consistent with our results. Since clone 28 recognized only a single epitope (adjacent to Tyr-530) and was specific for the nonphosphorylated, active form, the phosphorylation state of c-Src was more readily characterized with this antibody.
In addition to its usefulness in biochemical systems, we have also demonstrated the application of clone 28 in in vivo systems. In particular, Fig. 7clearly shows a restricted distribution of the active form in megakaryocytes, the progenitors of platelets. The tyrosine kinase activity of c-Src might be essential for platelet formation in the fetal liver, because c-Src was activated at all the developmental stages we observed (data not shown). Our data show good agreement with previous work, which reported high kinase activity in platelets by an in vitro kinase assay (Oda et al., 1992; Horvath et al., 1992; Clark and Brugge, 1993). In contrast to the intense staining in established cell lines such as 3Y1 (Fig. 6), immunohistochemical observations with mouse tissue sections revealed a restricted distribution even in the fetal stage, where cellular growth and differentiation actively occur (Fig. 7). These data, taken together, suggest that c-Src kinase activity would be suppressed in the steady state and activated transiently by extracellular stimuli in major tissues.
With clone 28, our immunofluorescence studies demonstrated distinctive staining of c-Src in normal rat cells, whereas with mAb 327, only a weak, diffuse staining pattern was observed. With mAb 327, it is very difficult to detect c-Src unless the protein is overexpressed by gene transfection (David-Pfeuty and Nouvian-Dooghe, 1990; David-Pfeuty et al., 1993; Kaplan et al., 1994). Our new mAb could provide valuable information about early events in carcinogenesis and other diseases in which Src family tyrosine kinases or associated SH2-containing proteins play a crucial role.