p135 src homology 2 domain-containing inositol 5'-phosphatase (SHIPbeta ) isoform can substitute for p145 SHIP in fcgamma RIIB1-mediated inhibitory signaling in B cells.

The inositol 5'-phosphatase, SHIP (also referred to as SHIP-1 or SHIPalpha), is expressed in all cells of the hematopoietic lineage. Depending on the cell type being investigated and the state of differentiation, SHIP isoforms of several different molecular masses (170, 160, 145, 135, 125, and 110 kDa) have been seen in immunoblots. However, the function of the individual isoforms and the effect of expressing multiple isoforms simultaneously are not understood. Some of these SHIP isoforms have recently been characterized at the level of primary sequence. In this report, we investigated the function of the recently characterized 135-kDa SHIP isoform (SHIPbeta), which appears to possess the catalytic domain but lacks some of the protein-protein interaction motifs at the C terminus. By reconstituting SHIP-deficient DT40 B cells with either SHIPbeta or the better-characterized p145 SHIPalpha, we addressed the function of SHIPbeta in the complete absence of SHIPalpha. We observed that SHIPbeta had enzymatic activity comparable with SHIPalpha and that SHIPbeta was able to reconstitute F(c)gammaRIIB1-mediated inhibition of B cell receptor-induced signaling events such as calcium flux and Akt and mitogen-activated protein kinase activation. SHIPbeta was readily phosphorylated in response to B cell receptor cross-linking with the inhibitory receptor F(c)gammaRIIB1 and SHIPbeta also interacted with the adapter protein Shc. During these studies we also observed that the SHIPalpha or SHIPbeta interaction with Grb2 is not required for F(c)gammaRIIB1-mediated inhibition of calcium flux. These data suggest that SHIPbeta, which is normally expressed in B cells along with SHIPalpha, functions comparably with SHIPalpha and that these two isoforms are not likely to be antagonistic in their function in vivo.

Essential role for the C-terminal noncatalytic region of SHIP in FcgammaRIIB1-mediated inhibitory signaling.

The inositol phosphatase SHIP binds to the FcgammaRIIB1 receptor and plays a critical role in FcgammaRIIB1-mediated inhibition of B-cell proliferation and immunoglobulin synthesis. The molecular details of SHIP function are not fully understood. While point mutations of the signature motifs in the inositol phosphatase domain abolish SHIP's ability to inhibit calcium flux in B cells, little is known about the function of the evolutionarily conserved, putative noncatalytic regions of SHIP in vivo. In this study, through a systematic mutagenesis approach, we identified the inositol phosphatase domain of SHIP between amino acids 400 and 866. Through reconstitution of a SHIP-deficient B-cell line with wild-type and mutant forms of SHIP, we demonstrate that the catalytic domain alone is not sufficient to mediate FcgammaRIIB1/SHIP-dependent inhibition of B-cell receptor signaling. Expression of a truncation mutant of SHIP that has intact phosphatase activity but lacks the last 190 amino acids showed that the noncatalytic region in the C terminus is essential for inhibitory signaling. Mutation of two tyrosines within this C-terminal region, previously identified as important in binding to Shc, showed a reduced inhibition of calcium flux. However, studies with an Shc-deficient B-cell line indicated that Shc-SHIP complex formation is not required and that other proteins that bind these tyrosines may be important in FcgammaRIIB1/SHIP-mediated calcium inhibition. Interestingly, membrane targeting of SHIP lacking the C terminus is able to restore this inhibition, suggesting a role for the C terminus in localization or stabilization of SHIP interaction at the membrane. Taken together, these data suggest that the noncatalytic carboxyl-terminal 190 amino acids of SHIP play a critical role in SHIP function in B cells and may play a similar role in several other receptor systems where SHIP functions as a negative regulator.

Roles of Lck, Syk and ZAP-70 tyrosine kinases in TCR-mediated phosphorylation of the adapter protein Shc.

The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, antigen receptors and cytokine receptors. Recent studies have suggested that tyrosine phosphorylation of Shc may play a key role in T lymphocyte proliferation via interaction of phosphorylated Shc with downstream molecules involved in activation of Ras and Myc proteins. However, the sites on Shc that are tyrosine phosphorylated in response to TCR engagement and the ability of different T cell tyrosine kinases to phosphorylate Shc have not been defined. In this report, we show that during TCR signaling, the tyrosines Y239, Y240 and Y317 of Shc are the primary sites of tyrosine phosphorylation. Mutation of all three tyrosines completely abolished tyrosine phosphorylation of Shc following TCR stimulation. Our data also suggest that multiple T cell tyrosine kinases contribute to tyrosine phosphorylation on Shc. In T cells, CD4/Lck-dependent tyrosine phosphorylation on Shc was markedly diminished when Y317 was mutated, suggesting a preference of Lck for the Y317 site. The syk-family kinases (Syk and ZAP-70) were able to phosphorylate the Y239 and Y240 sites, and less efficiently the Y317 site. Moreover, co-expression of Syk or ZAP-70 with Lck resulted in enhanced phosphorylation of Shc on all three sites, suggesting a synergy between the syk-family and scr-family kinases. Of the two potential Grb2 binding sites (Y239 and Y317), Y239 appears to play a greater role in recruiting Sos through Grb2. These studies have implications for Ras activation and mitogenic signaling during T cell activation.