EJECUCIÓN DE LA APLICACIÓN EN EL MINI-GRID

Phosphotyrosine-containing a2-SH 2 binding proteins

To detect tyrosine phosphorylated proteins binding a2-SH 2 domain, Triton X-100 soluble lysate of unstimulated and growth-factor treated PC12 cells were incubated with GST/SH2 fusion proteins of a2-chim aerin, Abl, Src, PLC-yl coupled to agarose beads; any associating proteins were detected by antiphosphotyrosine immunoblotting (see Methods 2.2.9).

GST/Src-SH2-SH3 bound an increased amount of phosphotyrosine proteins when PC12 cells were stimulated with EGF and NGF (Fig. 38). The presence of SH3 domain in the fusion protein may independently bind tyrosine-phosphorylated proteins or it may modulate the specificity of Src-SH2 domain (Weng et al., 1993). This co-operative effect has been observed for Lck-SH2-SH3 protein (Amrein et al., 1993). GST/Abl-SH2 also bound to a number of phosphotyrosine-containing proteins. In particular, it bound to a 45 kDa phosphoprotein specifically in EGF- and NGF- stimulated PC12 cells but not in control cells. In contrast, GST/(x2-SH2 had a faint band (80 kDa) from both unstimulated and growth factor-stimulated PC12 cells (Fig. 38). (The 65 kDa protein co-purified with GST/o2-SH2 during purification of the fusion protein (Fig. 27) and so it was not derived from P C I2 cells). GST/PLCyl-SH2, like GST/a2-SH2 did not bind a lot o f phosphoproteins. There was an increase in phosphorylation of a -1 7 0 kDa protein, which might be EGFR, binding to GST/PLCyl-SH2 in EGF-stimulated P C I2 cells. PLCyl-SH2 has been shown to bind phosphotyrosine-containing phosphoproteins such as Trk (Ohmichi et al., 1991) which migrated around 140 kDa. However, no 140 kDa protein was detected binding to PLCyl-SH2 from NGF-stimulated cells (Fig. 38). The difference in these observations could be due to the limit of detection for this method. Much more PC 12 cell proteins might be needed for detection of the interaction between PLC-yl and its target proteins while the same amount of PC 12 cells provided enough substrates for GST/Abl-SH2 and GST/Src-SH2-SH3. Thus, in PC 12 cells, a2-SH 2 interacted with specific tyrosine-phosphorylated substrates, whose abundance appeared to be less than those binding to Abl-SH2 domain or Src-SH2-SH3 domain.

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Fig. 38. SH2-domain associating proteins from PC12 cells as detected by

antiphosphotyrosine (PY20) immunoblotting. EGF and NGF were used at 100 ng/ml. The Triton soluble lysate was incubated with 20 pg of GST/SH2 fusion proteins coupled to glutathione agarose beads. (—) denotes

unstimulated cells. (*) indicates the position of the E.coli protein that

associated with GST/a2-SH2 fusion protein and cross-reacted with the antibody. ( ^ ) indicates a2-SH2 binding protein (80 kDa) and (►) indicates Abl-SH2 binding protein (45 kDa).

In antiphosphotyrosine immunoblotting, one problem arose with the use of GST-fusion protein non-covalently coupled to glutathione-agarose beads as an affinity matrix; this was the resultant interference from the GST fusion protein that was eluted together with the bound proteins. The mouse anti-phosphotyrosine antibody and several other antibodies cross-reacted with the GST fusion protein. The use o f proteins covalently bound to a solid matrix should circumvent the problem. The GST-SH2 fusion proteins of Abl and a2-chim aerin were therefore covalently coupled to CNBr-activated agarose beads (see Methods 2.2.2). The immobilised proteins were then used to isolate their associating proteins from PC12 cell lysates as described before. The covalently linked GST/a2-SH 2 bound more phosphotyrosine-containing proteins (Fig. 39) than its non-covalent linked counterpart (Fig. 38). The GST/a2-SH 2 binding proteins could be clearly detected, with a general increase in phosphotyrosine proteins binding cx2-SH2 at around 60- 100 kDa (Fig. 39). In contrast to previous results, GST/Abl-SH2 also bound the 45 kDa protein in unstimulated cells. The dissimilar results could be due to folding of SH2 domain being modified by covalent attachment o f some of its amino acids to the matrix support. In addition, the background (non-specific binding due to GST portion of the protein) from GST/SH2 fusion protein coupled to CNBr-activated Sepharose was also higher than when bound to glutathione agarose, especially when ^^P-labelled P C I2 ceU lysate was used. Nevertheless, the result showed that

GST/SH2 , fusion ^ NGF — 4- — — 4- kDa 97 - ' ' m -

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Fig. 39. PC 12 tyrosine-phosphorylated proteins binding to CNBr-coupled GST/SH2 beads detected by antiphosphotyrosine (PY20) Western immunoblotting. NGF was used at 100 ng/ml. The binding proteins were eluted with 1 M glycine, pH 2.5.

a2-SH2 binding protein in ^^P-labelled P C I2 cells

The interaction of a2-SH2 domain with phosphoproteins was further investigated with ^^P-labelled proteins from P C I2 cells (Fig. 40A) (see Methods 2.2.8). This method not only allowed the detection of low-abundance phosphoproteins but also those containing phosphoserine and phosphothreonine. PC 12 cells were stimulated with various growth factors such as NGF, aFGF, bFGF, EGF, tumor promoters PMA, cAMP and 20% serum (not all data shown). In all cases, similar phosphoproteins were associated with GST/a2-SH2. These included 38 kDa, 45 kDa, 60 kDa and 84 kDa proteins which were consistently observed but with varying intensity in the binding assays after treatment with different growth factors (Fig. 40 to Fig. 43). In particular, the 38 kDa protein bound specifically to GST/ot2-SH2. The phosphorylation of these proteins was slightly increased in response to NGF, aFGF and EGF (Fig. 40 to Fig. 42). More phosphoproteins bound

to GST/Src SH2-SH3 than to GST/a2-SH2 in NGF-stimulated PC12 cells which paralleled the observation from the antiphosphotyrosine immunoblot (Fig. 40B). In aFGF- and EGF-treated cells, a 45 kDa protein bound to GST/Abl-SH2 only in the stimulated cells and it could be the same protein detected by antiphosphotyrosine antibody (compare Fig.41 and Fig.42 with Fig.38). In addition, comparable levels of ^^P-labelled phosphoproteins bound to GST/Abl-SH2 and to GST/a2-SH2 (Fig. 41). This is in contrast to the observation from anti-phosphotyrsoine immunoblotting in which more phosphotyrosine proteins were shown to be binding to Abl-SH2 than to a2-SH2, indicating that most of the ^^P-labelled a2-SH2 binding proteins might not contain phosphotyrosine residues.

NGF — kDa 2 0 0 - 97 68 43 29 149

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