Src kinase inhibiter PP1 did not affect the G-CSF dependent tyrosine phosphorylation of Gab2
We have been studying the signal transductions of neutrophil precursor cells upon G-CSF stimulation. We found that Gab2 scaffold/adaptor protein was one of the major tyrosine phosphorylated proteins in neutrophil precursor cell line GM-I62-1 immediately after cells were stimulated with G-CSF. The resulted tyrosine phosphorylation of Gab2 activates downstream signaling pathways, including PI3K-Akt and SHP2-Ras-MAPK pathways, followed by the induction of various biological responses. Therefore, tyrosine phosphorylation of Gab2 is a crucial event on G-CSF dependent signal transduction reactions. However, a kinase which phosphorylates the tyrosine residues of Gab2 is not clearly defined. There is a report, in which pharmacological inhibitors, PD173 and PP1 inhibited the tyrosine phosphorylation of Gab2 upon G-CSF stimulation in murine pro-B-cell cell line Ba/F3 exogenously expressing G-CSF receptor, claiming that Src family kinase is responsible for the G-CSF dependent tyrosine phosphorylation of Gab2[14]. We examined the effects of Src kinase inhibitor, PP1, on the G-CSF stimulation dependent Lyn activation by immunoblot, using anti-p-Src family (Tyr416) antibody (Cell signaling technology, Danvers, MA), which reacts with the Src family kinases including Lyn when the tyrosine residue in the activation loop is phosphorylated. As shown in Fig. 1, Lyn was in an active state even after 4 hours of IL-3 starvation (Fig. 1 lane 1). Upon 5 min stimulation with G-CSF, the level of the activity of Lyn kept virtually unchanged (Fig. 1 lane 2). In the presence of Src kinase inhibitor, PP1 (10mM), the phosphorylation of the activation loop of Lyn was suppressed when cells were either unstimulated or stimulated with G-CSF (Fig. 1 lanes 3 and 4). Therefore, activation of Lyn was thoroughly inhibited in the presence of 10 mM PP1.
Then, the inhibitory effects of PP1 on the G-CSF dependent tyrosine phosphorylation of Gab2 was examined. As shown in Fig. 2 (lanes 1, 2, 7 and 8), G-CSF dependent tyrosine phosphorylation of Gab2 was not inhibited by the treatment of cells with 10mM PP1, although the activation of Lyn was markedly suppressed with the same concentration of PP1 (Fig. 1, lanes 3 and 4). Furthermore, another Src kinase inhibitor PP2 (3µM) did not inhibited the G-CSF stimulation dependent tyrosine phosphorylation of Gab2, either. SHP2 and p85 subunit of PI3K are known to bind to the specific tyrosine residues of Gab2, that is, Y604 and Y633 for SHP2 and Y441, Y465 and Y574 for p85 in mouse Gab2, in their phosphorylation dependent manner. As shown in Fig. 2, SHP2 and p85 were associated with Gab2 when Gab2 was tyrosine-phosphorylated by G-CSF stimulation (Fig. 2 lanes 1 and 2). The treatment with PP1 did not affect the binding of SHP2 and p85 to Gab2 (Fig. 2, lanes 7 and 8). These observations showed that Lyn was not involved in the G-CSF dependent tyrosine phosphorylation of Gab2 nor the phosphorylation-dependent association of SHP2 and p85 with Gab2 at least in the neutrophil precursor cell GM-I62-1.
Syk inhibitors Piceatannol and GS-9973 inhibited the G-CSF dependent tyrosine phosphorylation of Gab2
It was reported that Gab2 tyrosine phosphorylation was suppressed by the treatment of cells with pharmacological inhibitor against Syk, Piceatannol, or genetic manipulations of Syk in mast cell[26, 28]. Therefore, effects of inhibitors to the kinase, Piceatannol and GS-9973 (Entospletinib) were examined on the tyrosine phosphorylation of Gab2 upon G-CSF stimulation in the neutrophil precursor cells. As shown in Fig. 2, 30mM Piceatannol and 1mM GS-9973 suppressed the G-CSF dependent tyrosine phosphorylation of Gab2. Along with the decreased tyrosine phosphorylation of Gab2, the binding of SHP2 to Gab2 was reduced by the treatment of these inhibitors (Fig. 2, panel A and B lanes 1–6, Fig. 3, panel A and B). However, it is noteworthy that G-CSF dependent association of p85 subunit of PI3K with Gab2 was not affected by these inhibitors (Fig. 2 panel C lanes 1–6, Fig. 3, panel C). These observations indicated that tyrosine residues of Gab2 to which SHP2 binds were phosphorylated by GS-9973-sensiitive kinase, such as Syk, and that tyrosine residues to which p85 binds were phosphorylated by other kinase than Syk, which was insensitive to these inhibitors. Quantitative analysis of data from three independent experiments confirmed that Src kinase inhibitors, Piceatannol and GS-9973, inhibited the Gab2 tyrosine phosphorylation and SHP2 association to Gab2 (Fig. 3).
To confirm the inhibitory effects of GS-9973 on the tyrosine phosphorylation of Gab2 and the association of SHP2 with it, the G-CSF stimulation dependent tyrosine phosphorylation of Gab2 and its SHP2-association were re-examined when the cells were treated with increasing amounts of GS-9973. As shown in Fig. 4, the increasing inhibitory effects on the G-CSF dependent tyrosine phosphorylation of Gab2 and the association of SHP2 with Gab2 were observed when the cells were treated with increasing concentrations of GS-9973 (Fig. 4, A and B). Whereas the binding of p85 subunit of PI3K to Gab2 was not affected by the treatment of Syk inhibitor GS-9973 (Fig. 4, C). Furthermore, using another Syk inhibitor, Piceatannol, the concentration-dependent inhibitions of Gab2 tyrosine phosphorylation and the SHP2 binding to Gab2 were also observed (data not shown). However, the binding of p85 subunit to Gab2 was not affected by Piceatannol treatment (data not shown). These observations confirmed that the tyrosine residues of the SHP2 binding sites, Y604 and Y633 of Gab2 were phosphorylated by the GS-9973-sensitive kinase such as Syk. However, the tyrosine residues, Y441, Y465 and Y574 of Gab2 were phosphorylated by other kinase upon G-CSF stimulation, which is insensitive to GS-9973. Therefore, upon G-CSF stimulation, Gab2 tyrosine phosphorylation was caused by at least two distinct kinases, that is, GS-9973 sensitive one and insensitive one.