(Received for publication, October 5, 1995; and in revised form, November 29, 1995)
From the
A monoclonal antibody has been produced which immunoprecipitates 58- and 53-kDa proteins which are rapidly tyrosine phosphorylated in insulin-treated cells. These proteins can also be tyrosine phosphorylated in vitro by the isolated human insulin receptor. Increased tyrosine phosphorylation of these proteins is also observed in cells expressing a transforming chicken c-Src (mutant Phe-527) and in cells with the activated tyrosine kinase domains of the Drosophila insulin receptor, human insulin-like growth factor I receptor, and human insulin receptor-related receptor. P58/53 did not appear to associate with either the GTPase activating protein of Ras (called GAP) or the phosphatidylinositol 3-kinase by either co-immunoprecipitation experiments or in Far Westerns with the SH2 domains of these two proteins. Since p58/53 did not appear, by immunoblotting, to be related to any previously described tyrosine kinase substrate such as the SH2 containing proteins SHC and the tyrosine phosphatase Syp, the protein was purified in sufficient amounts to obtain peptide sequence. This sequence was utilized to isolate a cDNA clone that encodes a previously uncharacterized 53-kDa protein which, when expressed in mammalian cells, is tyrosine phosphorylated by the insulin receptor.
In the last few years, there has been extensive progress toward
an understanding of the mechanism whereby tyrosine kinases such as
growth factor receptors elicit subsequent biological
responses(1, 2) . The identification and isolation of
endogenous substrates for these molecules have revealed that various
enzymes such as phospholipase C are tyrosine phosphorylated and
activated by this modification(3) . In addition, other proteins
have a sequence which is homologous to a region of c-Src (called the
SH2 domain) that allows these proteins to bind to
tyrosine-phosphorylated proteins(4) . This complex formation
can itself stimulate the enzymatic activity of the SH2 containing
protein (for example, the binding of the phosphatidylinositol 3-kinase
to insulin receptor substrate-1) or redirect it to another cellular
compartment where its enzymatic activity is required (for example, the
translocation of the GTPase activating protein of Ras to the plasma
membrane, the site of its substrate)(4) . In the case of the
insulin receptor (IR), (
)several cytosolic substrates have
been described. These include the most extensively characterized
substrate, called insulin receptor substrate-1 (IRS-1), which is
tyrosine phosphorylated and subsequently bound by the
phosphatidylinositol (PI) 3-kinase as well as several other SH2
containing proteins(5) . A variety of experimental approaches
have implicated this substrate as playing a role in mediating several
biological responses (6, 7, 8, 9) although gene knockout
mice which lack IRS-1 still exhibit most of their responsiveness to
insulin(10, 11) . In addition, recent studies have
shown that growth hormone, interleukins 4 and 13, interferons-
and
, and leukemia inhibitory factor can all stimulate the tyrosine
phosphorylation of IRS-1 (12, 13, 14) .
In addition to IRS-1, a number of other tyrosine-phosphorylated proteins have been observed after insulin-stimulation of cells. These include at least one 115-kDa protein which has been found to associate with the SH2 containing tyrosine phosphatase Syp (15, 16) and two 60-kDa proteins which have been found to associate with various SH2 containing proteins including the GTPase activating protein of Ras (called GAP) and the phosphatidylinositol 3-kinase(17, 18, 19, 20) . These two 60-kDa proteins appear to be distinct since some cells, such as adipocytes, contain predominantly the PI 3-kinase associated 60-kDa protein(17) , whereas other cells, such as Chinese hamster ovary cells (CHO), contain predominantly the GAP-associated protein(19) . At least one cell type (the rat hepatoma called HTC) appears to contain both of these proteins(20) . In prior studies a monoclonal antibody has been generated against the tyrosine-phosphorylated 60-kDa GAP-associated protein in CHO cells and this antibody was found not to recognize the PI 3-kinase-associated 60-kDa protein, further indicating that these two proteins are distinct(19) . In the present studies we have therefore sought to produce a monoclonal antibody that recognizes the PI 3-kinase-associated 60-kDa protein. A monoclonal antibody was generated against a 60-kDa tyrosine phosphorylated substrate from the HTC cells. However, this antibody was found to recognize a protein that appeared to be distinct from both the GAP and PI 3-kinase-associated 60-kDa tyrosine phosphorylated proteins. The sequence of this protein revealed that it was indeed unique and thus represents a new substrate for tyrosine kinases such as the IR.
The eluates from 6 preparations were injected over a 2-week period into the hind foot pads of two Balb/c mice and the lymphocytes from the draining lymph nodes were fused to myeloma SP2/O cells using polyethylene glycol, seeded into 24-well plates, and selected with hypoxanthine-azaserine (Sigma)(19) . To screen the resulting hybridomas, their supernatants were incubated with rabbit anti-mouse IgG-coated protein A-agarose and the adsorbed antibodies were tested for the ability to immunoprecipitate tyrosine-phosphorylated proteins from insulin-treated HTC-IR cell lysates. Positive hybridomas were identified and cloned by serial dilution.
GST fusion protein blotting was carried out as described by Kuhne et al.(25) with minor modifications. After transfer, the membranes were blocked with HBS containing 5% dry milk and 0.05% Tween 20 and then incubated with a mixture of GST fusion protein (20 ng/ml) and affinity purified polyclonal anti-GST antibody (1 µg/ml) in HBS containing 1% dry milk and 0.05% Tween 20 and then visualized with horseradish peroxidase-conjugated secondary antibodies and ECL.
The cloned 600-bp PCR product was radiolabeled using
[-
P]dCTP (Amersham) and the multiprime DNA
labeling system (Amersham) and used to screen an oligo(dT)-primed
Uni-ZAP XR CHO cDNA library (Stratagene) as well as a random and
oligo(dT)-primed
gt11 CHO cDNA library (Clontech). Positive clones
were subcloned into pBluescript and sequenced with the U. S.
Biochemical Corp. Sequenase 2.0 kit (Amersham). A full-length clone was
obtained by replacing the incomplete 5` end of the largest Uni-ZAP
clone, an EcoRI-AatII fragment, with the 5` end of
one of the
gt11 clones. The full-length p53 cDNA was sequenced on
both strands and the sequences were analyzed using IntelliGenetics and
BLAST. Both the non-redundant and expressed sequence tag data bases
were searched.
Figure 1: Identification of a monoclonal antibody to p58/53. HTC-IR cells were treated with 0.5 mM vanadate and 1 µM insulin, lysed and the lysates were immunoprecipitated with either a polyclonal antibody to the 85-kDa subunit of PI 3-kinase (p85), a monoclonal antibody to the 60-kDa GAP-associated protein (p60), the new monoclonal antibody called H720, or control mouse immunoglobulin (NMG). The immunoprecipitates were analyzed by SDS-PAGE and immunoblotted with either anti-phosphotyrosine antibodies (anti-ptyr) or H720. Positions of prestained protein markers (in kDa) are indicated.
To further test this hypothesis, p58/53 and
the two 60-kDa tyrosine-phosphorylated proteins were immunoprecipitated
from insulin-treated HTC-IR or CHO.T cells, electrophoresed on SDS
gels, transferred to nitrocellulose, and blotted with either the SH2
domains of GAP or the 85-kDa subunit of PI 3-kinase or the SH3 domain
of GAP. As expected, the GAP-associated p60 was recognized by the SH2
domain of GAP whereas the PI 3-kinase-associated 60-kDa protein was
specifically recognized by the SH2 domain of the 85-kDa subunit of PI
3-kinase (Fig. 2), further indicating the distinct nature of
these two proteins(19) . In contrast, p58/53 was not recognized
by either SH2 domain nor by the SH3 domain of GAP (Fig. 2). In
other experiments, the SH2 domains of abl, phospholipase C, Syp,
Grb2, Src, and SHC were also found not to bind to
tyrosine-phosphorylated p58/53 (data not shown). Further evidence for
the distinct nature of p58/53 and the two 60-kDa proteins came from
studies showing that the H720 antibodies did not immunoprecipitate
either GAP or the PI 3-kinase in association with p58/53 (data not
shown). Finally, p58/53 could be depleted from cell lysates with
antibody H720 without affecting the levels of the GAP-associated p60
(data not shown). All of these data argue for the distinct nature of
p58/53.
Figure 2: Inability of tyrosine-phosphorylated p58/53 to be recognized by either the SH2 domains of GAP or p85 or by the SH3 domain of GAP. The 60-kDa tyrosine-phosphorylated PI 3-kinase-associated protein (immunoprecipitated from HTC-IR cells with antibodies to p85), the GAP-associated protein (immunoprecipitated from insulin-treated CHO.T cells with the p60 antibody), and p58/53 (immunoprecipitated from insulin and vanadate-treated CHO.T cells with monoclonal antibody H720) were analyzed by SDS-PAGE and blotted with anti-phosphotyrosine antibodies (anti-ptyr), the SH2 domains of either GAP or p85 or the SH3 domain of GAP.
Figure 3: Effect of IR overexpression and vanadate on the insulin-stimulated tyrosine phosphorylation of p58/53. CHO or CHO.T cells were incubated with or without 0.5 mM vanadate for 15 min and then stimulated with 1 µM insulin for 7 min, as indicated. Cell lysates were immunoprecipitated with H720 and the bound proteins were eluted and analyzed by SDS-PAGE and Western blotting with anti-phosphotyrosine antibodies.
Figure 4: In vivo and in vitro phosphorylation of p58/53. A, time course. CHO.T cells were treated with 0.5 mM vanadate for 30 min and then stimulated with 1 µM insulin for the times indicated. The cells were then lysed and processed as described in the legend to Fig. 3. B, dose curve. CHO.T cells were treated with 0.5 mM vanadate for 30 min and then stimulated with the indicated concentrations of insulin for 7 min, lysed, and processed as above. C, in vitro phosphorylation of p58/53. CHO cell lysates were immunoprecipitated with either monoclonal antibody H720 (p58) or control immunoglobulin (N), and the immunoprecipitates were incubated with or without isolated IR in the presence of ATP as described under ``Experimental Procedures.'' The immunoprecipitates were then analyzed by SDS-PAGE and immunoblotting with anti-phosphotyrosine antibodies.
To test whether p58/53 were substrates of other tyrosine kinases, we first examined the ability of other members of the IR family to phosphorylate these proteins. In CHO-PKC cells overexpressing either the human IGF-I receptor or chimeric receptors with the cytoplasmic domains of the Drosophila IR or the human insulin receptor-related receptor and the extracellular domain of the IR, insulin was found to stimulate the tyrosine phosphorylation of p58/53 (Fig. 5). In contrast, in the parental cells, no insulin-stimulated tyrosine phosphorylation of these proteins was observed, indicating that the insulin-stimulated increase in tyrosine phosphorylation required the presence of these expressed receptors. Since in prior studies activation of PKC was found to inhibit the in situ tyrosine phosphorylation of several other substrates of the various members of the IR family(24) , we also tested whether the pretreatment of these cells with an activator of PKC, phorbol 12-myristate 13-acetate, affected the ability of these receptors to tyrosine phosphorylate p58/53. In each case, PKC activation inhibited the insulin-stimulated tyrosine phosphorylation of p58/53 (Fig. 5). In studies of other cells, neither epidermal growth factor, nerve growth factor, nor platelet-derived growth factor were found to stimulate an increase in tyrosine phosphorylation of p58/53 although p58/53 was found to have increased tyrosine phosphorylation in cells expressing a transforming c-Src (the F527 mutant) (data not shown). Since this Src has previously been shown to activate endogenous IGF-I receptors(26) , it is possible that the increased phosphorylation of p58/53 is mediated via this receptor.
Figure 5:
Tyrosine phosphorylation of p58/53 by
other members of the IR family and inhibition of this phosphorylation
by activation of PKC. CHO-PKC
cells overexpressing either the
human IGF-I receptor (CHO-PKC-hIGFR), a chimeric receptor with the
extracellular domain of the human IR and the cytoplasmic domain of the
human insulin receptor related-receptor (CHO-PKC-hIR/hIRR), or the
cytoplasmic domain of the Drosophila IR (CHO-PKC-hIR/dIR) or
the parental cells only overexpressing PKC (CHO-PKC) were stimulated
with ligand as indicated. Some cells were also treated with 1
µM phorbol 12-myristate 13-acetate (PMA) for 20
min prior to the ligand stimulation, as indicated. The cells were lysed
and the immunoprecipitated p58/53 was analyzed by SDS-PAGE and
immunoblotting with anti-phosphotyrosine
antibodies.
To determine whether p58/53 was associated with other proteins in the cell, CHO.T cells were metabolically labeled with radioactive methionine and cysteine, treated with or without insulin, lysed in a homogenizer in the absence of detergent and the lysates were separated into either a particulate or soluble fraction. After solubilization of the particulate fraction with detergent, the two fractions were immunoprecipitated with either the H720 antibody or control immunoglobulin. In addition, p58/53 was also immunoprecipitated from Triton-solubilized total lysates of metabolically labeled CHO.T cells. The immunoprecipitates were analyzed by SDS-PAGE and autoradiography. The H720 immunoprecipitates contained labeled 58- and 53-kDa proteins and a small amount of a 45-kDa band (Fig. 6). Exactly the same pattern was observed if the cells were treated with insulin (data not shown). A small amount of a tyrosine-phosphorylated 45-kDa band was also observed in p58 precipitates from CHO.T cells ( Fig. 2and Fig. 4), suggesting that this lower molecular weight band may be either a proteolytic fragment of p58/53 or an alternate form of this protein. Interestingly, both p58 and p53 (as well as the 45-kDa band) appear to be primarily present in the particulate fraction of the lysate (Fig. 6). This location of the protein was also unaffected by the addition of insulin to the intact cells prior to lysis (data not shown).
Figure 6:
Immunoprecipitation of p58/53 from
metabolically labeled cells. CHO.T cells were labeled with
[S]methionine and cysteine, lysed by
homogenization, and the lysates were fractionated into a cytosolic (sup) and particulate (part) fraction as described
under ``Experimental Procedures.'' Each fraction was
immunoprecipitated with either antibodies to p58/53 (p58) or
control immunoglobulin (N) and the immunoprecipitates were
analyzed by SDS-PAGE and autoradiography. Immunoprecipitations were
also performed from the total cell lysates (total).
To determine the tissue distribution of p58/53, lysates of different mouse organs were immunoprecipitated with H720 and the immunoprecipitates were phosphorylated in vitro with isolated IR. For comparison, the lysates were also immunoprecipitated with the antibody to the GAP-associated 60-kDa protein and these immunoprecipitates were also phosphorylated in vitro with the isolated IR. Of the tissues tested (brain, spleen, muscle, and liver), p58/53 was found to be highest in brain (Fig. 7). In contrast, the GAP-associated p60 was found to be most abundant in spleen (Fig. 7), further indicating the distinct nature of these proteins.
Figure 7: Tissue distribution of p58/53 and the GAP-associated p60. CHO.T cells or the indicated mouse tissues were lysed and comparable amounts of protein from each tissue were immunoprecipitated with either antibodies to the GAP-associated 60-kDa tyrosine-phosphorylated protein (p60), antibodies to p58/53 (p58), or control immunoglobulin (N). The immunoprecipitates were then phosphorylated in vitro with isolated IR and analyzed by SDS-PAGE and immunoblotting with anti-phosphotyrosine.
In addition to the GAP and PI 3-kinase-associated 60-kDa tyrosine-phosphorylated proteins, at least two other substrates of tyrosine kinases have been described which also have molecular masses close to p58. These include the tyrosine phosphatase Syp (27) and the linker protein SHC(28) . Immunoprecipitates with antibodies to these two proteins showed that their tyrosine phosphorylation patterns were quite distinct from that of p58/53 and that the p58/53 proteins immunoprecipitated by monoclonal antibody H720 did not react with antibodies to either of these two proteins (Fig. 8). Thus, these studies suggested that p58/53 is a previously uncharacterized substrate for tyrosine kinases and further indicates that it does not associate with either of these two SH2 containing proteins.
Figure 8: Lack of immunoreactivity of p58/53 with anti-SHC and anti-Syp antibodies. Insulin-treated CHO.T cells were lysed and immunoprecipitated with either the monoclonal antibody to p58/53, a polyclonal antibody to Syp, a polyclonal antibody to SHC, or control immunoglobulin (NMG). The immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with either anti-phosphotyrosine antibodies (anti-ptyr), antibodies to SHC or Syp, as indicated.
Figure 9: Nucleotide sequence and deduced amino acid sequence of p53. The amino acid sequences of the two tryptic peptides are underlined, the only difference being at residue 78 where glycine, instead of glutamine, was present in the initial tryptic peptide sequence.
To test whether the protein product of the isolated cDNA was recognized by the monoclonal antibody H720, the cDNA was subcloned into the mammalian expression vector pCLDN and either this plasmid or the parental vector was transiently transfected into COS cells. The monoclonal antibody to p58/53 specifically immunoprecipitated a single H720 immunoreactive band from COS cells transfected with the isolated cDNA but not from COS cells transfected with the vector control (Fig. 10). To test whether the expressed protein could be tyrosine phosphorylated, the plasmids were transfected into either CHO.T cells or into COS cells either with or without a plasmid encoding the IR(23) . The cells were treated with insulin and vanadate, lysed, and H720 immunoprecipitates from the lysates were immunoblotted with anti-phosphotyrosine antibodies. In the CHO.T cells transfected with the isolated cDNA, an increase in the tyrosine phosphorylation of only the p53 band was observed over that seen in the control cells transfected with the vector alone (Fig. 10). In the COS cells transfected with only the isolated cDNA, a single tyrosine-phosphorylated band was observed that co-migrated with the lower (p53) band in CHO.T cells (Fig. 10). In COS cells transfected with only the IR encoding plasmid, the tyrosine phosphorylation of the endogenous p58/53 was observed. In contrast, in the cells transfected with both the IR and the p53 plasmid, a much greater increase in the tyrosine phosphorylation of the p53 band was observed compared to the cells transfected with either plasmid alone (Fig. 10). In other studies, insulin treatment was found to stimulate a 2-3-fold increase in the tyrosine phosphorylation of p53 in COS cells transiently transfected with the isolated cDNA and the IR. These results indicate that the isolated cDNA encodes the 53-kDa tyrosine-phosphorylated band observed in CHO.T cells. Since the peptide sequence utilized for isolating this cDNA came from p58, these results further support the hypothesis that p58 and p53 are highly related, most likely due to alternative splicing of the mRNA or possibly due to post-translational modifications. The relative levels of these two different forms of the protein differed in various cells and tissues, with HTC cells having predominantly p58 whereas brain had predominantly p53.
Figure 10: Transient expression of p53 and its tyrosine phosphorylation by IR. CHO.T or COS-7 cells were transiently transfected with control pCLDN vector (V), pCLDN containing the cloned cDNA (p53), and/or a plasmid encoding the IR (IR). P58/53 was immunoprecipitated from the cell lysates and analyzed by SDS-PAGE and immunoblotting with either the monoclonal antibody to p58/53 or anti-phosphotyrosine (anti-ptyr). For the tyrosine phosphorylation experiments, the cells were stimulated with 0.5 mM vanadate and 1 µM insulin prior to lysis.
In the present studies, a monoclonal antibody has been produced against 58/53 kDa proteins which rapidly become tyrosine phosphorylated in CHO.T and HTC-IR cells treated with insulin. Although this molecular mass is close to several previously described substrates for tyrosine kinases including a 60-kDa GAP-associated and a 60-kDa PI 3-kinase-associated protein, the 58/53-kDa proteins did not appear to be either of these two substrates since they did not associate with either GAP or PI 3-kinase in vitro or in vivo. Also, the monoclonal antibody to p58/53 did not react on Western blots with either of these two other substrates. The purification of the p58/53-kDa proteins allowed us to obtain the sequence of two peptides from p58 which was used to obtain a cDNA which encodes for p53. The deduced sequence of p53 indicates that this protein is a previously unidentified substrate for tyrosine kinases. Interestingly, this protein appears to be primarily in the particulate fraction of cell lysates, possibly suggesting that this protein is either membrane-associated or in a particular subcellular compartment. In this regard, it is of interest that the carboxyl-terminal 3 amino acids (A-R-F) of p53 fits a consensus sequence ((SAGCN)-(RKH)-(LIVMAF)) for targeting to peroxisomes although this is a relatively uncommonly utilized combination of amino acids(31) . Also, a stretch of 46 amino acids in the carboxy tail of p53 (residues 408 to 454) were identified by BLAST and MOST as exhibiting homology with the SH3 domains of 2 yeast proteins, called BOB1 and BEB1(32, 33) . The presence of a possible SH3 domain in p53 would also be consistent with its location in the particulate fraction of cells since this motif is often found in cytoskeletal-associated proteins and would suggest a role for this protein in cell signaling(34) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U41899[GenBank].