Results

Binding affinities of FcyRIIb, SHIP and She phosphopeptides to the recombinant SH2 domains ofSHP-1, SHP-2 and SHIP

We have analysed the interactions between the phosphopeptides listed in Table I., and recombinant proteins consisting of the SH2 domains of SHP-1, SHP-2 and SHIP, respectively, by using surface plasmon resonance (SPR). The phosphopeptides were biotinylated and bound to the streptavidin coated biosensor chips. Typical association and dissociation curves are shown on Fig.l. Both dimeric SH2 domains of SHP-2 and monomeric SH2 of SHIP bound the p-ITIM peptide with high affinity (K

=104xl0"

M, and 40x10"

M, respectively). In contrast, SHP-1 SH2 domains exhibited rather low affinity to p-ITIM (K

=10"3 M) (Fig 1. a) and did not bind to any of the other phosphopeptides tested. SHP-2 and SHIP SH2 domains bound to all phosphopeptides, with apparent Kp of 60-305x10"

M and 14-145xl0~

M, respectively (Fig. lb, lc). The K^ values calculated from the kinetic measurements are summarised in Table II. None of the SH2 domains reacted with tyrosine phosphorylated peptides corresponding to the PI3-K binding consensus sequences of CD 19.

In order to establish a model for studying the possible interaction of the phosphorylated FcyRIIb-SHIP complexes with SHP-2 a bisphosphopeptide was synthesised, combining FcyRIIb p-ITIM with the N-terminal phosphopeptide of SHIP by a 50 A long flexible linker consisting of 15 A-G dimer repeats. This SHIP-p-ITIM peptide is potentially capable to engage both SH2 domains of the recombinant SHP-2 protein. Indeed, SPR measurements of the interaction between this compound and SPIP-2 SH2 domains established an apparent K

=9xl0"

M, i.e. an approximately 10 times higher affinity constant than that of the p-ITIM for these domains and a 30 times increase as compared to the K

of the

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corresponding SHIP peptide. Examining closer the association-dissociation curves, a considerably higher K

rate was observed between the bisphosphopeptide and SHP-2, as compared to the K

between the monophosphopeptides and SHP-2 (Table 2., Fig. Id). SHP-2 and SHIP bound to the bisphosphopeptide with a similar affinity.

Activation of SHP-2 by the SHIP -p-ITIM bisphosphopeptide

Catalytic activity of SHP-2 has been shown to increase dramatically upon occupancy of both its SH2 domains by bisphosphorylated tyrosine-based motifs. This was rationalised by the disruption of the interaction between SHP-2 phosphatase domain and the N-terminal SH2 domain (37, 38). We have shown previously that the p-ITIM peptide bound to an insoluble matrix precipitated from BL41 cell lysates considerable tyrosine phosphatase activity (21,22, 23). The catalytic activity of immunoisolated SHP-2 was now tested on different phosphopeptides substrates by ELI SA measurements of their remaining phosphotyrosine content. The immunoprecipitated SHP-2 samples exhibited a single protein band corresponding to SHP-2 by Western blot detection. This band was not recognised by SHP-1 specific antibody. Comparing the phosphotyrosine content of the non-treated phosphopeptide samples with those treated with various dilutions of SHP-2, 60% dephosphorylation of

SHIP-«

p-ITIM bisphosphopeptide was obtained by as high as a 70 fold dilution of SHP-2 immunoisolated from 2xl0

BL41 cells-equivalent lysate. A 10 and 20 fold dilution was required to dephosphorylate the various monophosphopeptides at the same rate. Similar differences were observed comparing SHP-2 dilutions required for 20% dephosphorylation of the phosphopeptides (Fig.2). These results indicate that the SHIP-p-ITIM bisphosphopeptide significantly enhances the catalytic activity of SHP-2.

Binding of Grb2 and Gabl to the phosphopeptides of FcyRIIb, SHIP and She

Recently the tyrosine phosphorylated Grb2-binding, docking protein, Gabl was

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reported to play a role in translocation of the SH2 domain containing SHP-2, PI3-K, and She to the cell membrane of activated B cells (10). We have tested the binding from BL41 cell lysate samples of Gabl and Gab 1-associated molecules to p-ITIM and to phosphopeptides identical with the tyrosine phosphorylated motifs of p-ITIM-recruited proteins (Fig.3). As controls, tyrosine phosphorylated peptides corresponding to the CD 19 C-terminal sequence were used. Both CD 19 phosphopeptides bound PI3-K (data not shown), but did not react with any other SH2 domain containing signaling molecules, such as SHP-2, SHIP, She, and Grb2.

FcyRIIb p-ITIM, SHIP-p-ITIM bisphopshopeptide and the C- terminal phosphopeptides of SHIP bound She, while SHIP-p-ITIM and the N-terminal phosphopeptide of SHIP, as well as both the C- and N-terminal She peptides bound Grb-2. Binding of Grb-2 to p-ITIM was also observed after longer incubation times. Pre-clearance immunoprecipitation by 2 pg Gabl specific antibodies /5*10

cells considerably reduced She and Grb2 binding to SHIP-p-ITIM (Fig.3a).

The binding of SHP-2 and Grb2 to the bisphosphopeptide, SHIP-p-ITIM was also tested in a competition experiment. Only the p-ITIM peptide decreased the binding of SHP-2 to SHIP-p-ITIM, while the N-terminal SHIP and She phosphopeptides competed for Grb-2 binding (Fig.3b). Thus binding of Grb2 to p-ITIM may be indirect.

Since SHP-2, She, and Grb2 were detected in samples precipitated by the p-ITIM and SHIP-ITIM phosphopeptides from cell lysates, we tested whether Gabl is present in the p-ITIM bound complex. Though Gabl lacks an SH2 or phosphotyrosine binding (PTB) domains, it did bind to both p-ITIM, and SHIP-p-ITIM phosphopeptides. Moreover, it reacted with the N-terminal phosphopeptide of SHIP and with the C-terminal phosphopeptide of She (Fig.3c). These results indicate that the binding of Gabl to these tyrosine phosphorylated peptides is indirect, probably mediated by molecules with multiple SH2 or

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PTB domains.

Interaction between Gabl and SHP-2

In order to examine the interaction between Gabl and SHP-2, we have synthesised three phosphopeptides, two of which corresponded to the assumed SHP-2 binding motifs of Gabl (Ingham et al. 1998)). Binding of these phosphopeptides to PI3-K was also monitored and compared with that of p-ITIM. All three phosphopeptides bound SHP-2, while only the N-terminal phosphopeptide (Q - C), containing the YLLL motif bound both PI3-K and SHP-2. The p-ITIM peptide bound SHP-2 most efficiently. The peptide containing the phosphorylated tyrosine 626 (YLDL) was a strong SHP-2 binder, the C-terminal phosphopeptide (YVVV) bound weakest, while the N-terminal phosphopeptide showed intermediate binding

Q-

C (Fig.4a). The level of tyrosine phosphorylation was similar in all peptides as controled by ELISA.

The phosphopeptides corresponding to three tyrosine phosphorylation motifs of Gabl were also compared for their SHP-2 activation capacity, which was found to correlate with their SHP-2 binding. One of them (

GDQVEYLDLDLD

) was the most efficient one as evidenced by its complete dephosphorylation (Fig. 4b).

FcyRIIb-BCR co-clustering induces Gabl dephosphorylation and its dissociation from PI3-K FcyRIIb and Gabl were affinity purified from untreated, BCR activated or BCR-FcyRIIb co-clustered BL41 cells. The isolated proteins were analysed after SDS-PAGE by immunoblotting. Both Gabl and Grb2 were detected in FcyRIIb precipitates obtained from BCR clustered and FcyRIIb-BCR co-clustered cells after 2 min treatment, while the untreated control samples and cells treated only for 30 sec were negative (Fig.Sa).

In order to examine the influence of BCR-FcyRIIb co-aggregation on the state of Gabl tyrosine phosphorylation, affinity purified Gabl samples immunoisolated from resting

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cells, or from BCR clustered and BCR-FcyRIIb co-clustered cells after 5 min treatment were separated by electrophoresis and immunoblotted with phosphotyrosine specific antibodies.

Tyrosine phosphorylation of the 110 kDa protein corresponding to Gabl significantly decreased upon co-clustering FcyRIIb and BCR, as compared to the BCR activated samples, indicating that Gabl undergoes dephosphorylation (Fig.5b, left panel).

Association of Gabl with SHP-2 was tested by analysing affinity purified SHP-2 samples obtained from resting, BCR-activated and BCR-FcyRIIb co-clustered BL41 cells.

The level of bound Gabl was the same in the latter two samples, indicating that co-clustering of antigen and FcyRIIb receptors on human B cells does not influence the Gabl association with SHP-2 induced by BCR clustering (Fig. 5b, right panel).

To asses whether the tyrosine phosphorylation state of Gabl affects its binding to PI3-K, this interaction was tested in immunoisolated PI3-K samples. BCR-FcyRIIb co-aggregation abolished the association between the p85 subunit of PI3K and Gabl, as monitored by both phosphotyrosine and Gabl specific antibodies. The same result was obtained in the reverse experiment, we could not observe PI3-K in the immunoisolated Gabl samples obtained from FcyRIIb-BCR co-clustered cells (data not shown). Thus co-clustering of the BCR and FcyRIIb induces dephosphorylation of Gabl and the dissociation of p85 PI3-K from Gabl (Fig. 5c).

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