fr SIPS clip PlPs Multiple
of an inositol
KAVANAUCH, W. M. et al. (1996) polyphosphate 5phosphatase form signaling Cut-r. Biol. 6, 4381145
Receptor and non-receptor tyrosine kinases coordinate the formation of signal-transduction complexes. Interactions between intracellular signalling proteins rely on the presence of phosphotyrosine-binding motifs [the Src homology 2 (SH2) and PTB domains] and the commonly-associated SH3 domain (which binds to prolinerich stretches of amino acids). Identifying new members of such signalling complexes is of obvious importance, especially if they turn out to have an interesting enzymatic activity. Here, Kavanaugh and colleagues have identified a new family of enzymes - the SIPS -which interact with two prominent signalling proteins, She and Crb2. From a human cDNA expression library, proteins were sought that could interact with Grb2 specifically via its SH3 domains, but not with other SH3containing proteins. One such clone was identified and found to encode a protein of predicted mass 110 kDa, having extensive homology to the family of enzymes that act as inositol
VAUGHAN, T. J. et al. (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library Nature Biotechnology 14, 309-314 Human monoclonal antibodies (mAbs) are an invaluable research tool for the cell biologist as well as having great therapeutic potential. Previously, the generation of human mAbs by phage display had not reached its full potential. Although highly specific mAbs were readily isolated, they were generally of moderate-to-low affinity (pm range), which is probably a function of library size (
in CELL BIOLOGY
6) July 1996
polyphosphate 5phosphatases (IF’s). This ~110 possesses several prolinerich (SH3-binding) motifs, and furthermore, epitope-tagged versions of pII0 and Grb2 interact in vivo. This has led to pl IO being termed a signalling IF’, or SIP, because of its connection with signal-transduction proteins. To search for proteins that bind to She, via its PTB domain, a protein purification strategy (rather than expression cloning) was used, and, surprisingly, this revealed two further members of the SIP family, SIP1 30 and SIP1 45. These two proteins formed a stable complex with She in stimulated (but not resting) B cells, whereas SIP1 10 did not. All three SIPS are encoded by the same gene, with alternative translation giving rise to SIP1 30 and SIP145, and alternative splicing accounting for the smaller SIP1 10. The N-terminal extension present in the two larger SIPS presumably accounts for their interaction with the PTB domain of She. Interestingly, this part of SIP1 30 and SIP1 45 also houses an SH2
domain, giving them the potential to interact with additional signalling proteins. Are the SIPS phosphatases? SIP1 10 was confirmed as having inositol polyphosphate 5-phosphatase activity, being able to hydrolyse both inositol (1,3,4,5)-tetrakisphosphate and phosphatidylinositol(3,4,5)-trisphosphate. SIP1 30 and/or SIP145 are very likely to have similar properties, since such phosphatase activity also associated with She in stimulated B cells. The 5-phosphatase activities showed a characteristic preference for substrates phosphorylated in the 3-position of the inositol ring, providing a possible link to the phosphoinositide (PI) 3-kinase signalling pathway. One possibility is that the SIPS represent a negative-feedback mechanism to control the activity of the PI 3-kinase pathway downstream of activated receptors, just as the GTPase-activating protein (GAP) engaged by these receptors may regulate the activity of the Ras pathway.
The affinities of such s&s match those of mAbs produced by traditional hybridomas, yet they take only two weeks to isolate. It seems likely, therefore, that large phage libraries of antibody genes may soon supersede traditional hybridoma technology.
out mainly by the 205 and 26s proteosomes in the cytosol. In this paper, Groettrup and co-workers present evidence that an interferon-inducible activator of 205 proteasomes, PA28, plays a role in this process. Groettrup and colleagues expressed enhanced levels of the PA28 CI subunit in mouse fibroblast cells containing the murine cytomegalovirus pp89 protein. They found that an increase in the level of PA28a markedly increased the efficiency of recognition by cytotoxic T cells specific for a pp89 peptide, as measured by lysis of the fibroblast cells. A similar effect on presentation of an influenza nucleoprotein peptide was also observed. These results provide further evidence for an important role of 205 proteasomes in antigen presentation, and suggest an additional means by which interferon could regulate antigen presentation (it also alters the subunit composition of the proteasomes). The mechanism of PA28 activation is not yet known, but in vitro data has suggested that it affects both the specificity and rate of peptide generation by 205 proteasomes.
TAn interfering activator CROETTRUP, C. et al. (1996) A role for the proteasome regulator PA28a in antigen presentation Nature 381, 166-l 68 Protein antigens are recognized by cytotoxic T cells as short peptides presented on the cell surface bound to MHC class I molecules. Degradation of proteins to short peptides is an important aspect of the antigen-presentation process, and is thought to be carried
This month’s headlines were contributed by Debbie Sweet, Peter Thomason and Dawn Walters. 259