Abstract
CD22 phosphorylation is an early event of B cell antigen receptor engagement and results in the recruitment of the negative regulatory tyrosine phosphatase, SHP-1. Peptides representing the potential phosphorylation sites within the cytoplasmic domain of CD22 have been used to stimulate SHP-1 catalytic activity and to inhibit the binding of SHP-1 to CD22 (Doody, G., Justement, L., Delibrias, C., Matthews, R., Lin, J., Thomas, M., and Fearon, D. (1995) Science 269, 242-244). However, the sites of phosphorylation within the cytoplasmic domain of CD22 and the importance of each for the recruitment and activation of SHP-1 remain unknown. Here we demonstrate that there are multiple sites within the cytoplasmic domain of CD22 that interact with the Src homology 2 domains of SHP-1. Nevertheless, a minimum of two tyrosines in CD22 is required for the association with SHP-1. Furthermore, both Src homology 2 domains of SHP-1 are necessary for efficient binding to CD22.
Highlights
Engagement of the B cell antigen receptor (BCR)1 results in an increase in tyrosine phosphorylation
SHP-1 with a single functional Src homology 2 (SH2) domain was capable of binding the CD22 cytoplasmic domain; the binding was dramatically decreased. Both SH2 domains of SHP-1 are required for efficient binding to the cytoplasmic domain of CD22
We demonstrate that at least two functional immunoreceptor tyrosine-based inhibitory motif (ITIM) and both SH2 domains of SHP-1 are required for efficient binding to the cytoplasmic domain of CD22
Summary
Cell Lines and Antibodies—HeLa cells were obtained from the ATCC (Rockville, MD) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% bovine calf serum and L-glutamine. Mutations were introduced into the SH2 domains of SHP-1 by polymerase chain reaction (PCR) site overlap extension as described.. To construct SHP-1(⌬P), PCR site overlap extension was used to generate a BglII-BalI fragment with a deletion of the phosphatase catalytic domain signature motif (451VHCSAG456); the PCR primers used were 5Ј-CACAGCAGAATACAAACTGC-3Ј (forward, outer), 5Ј-TCATGCAGGGCCCATCATTACGGGTACC-3Ј (forward, inner), 5ЈTATCCAATGACGATGATGGTACCCGTAATGATGGG-3Ј (reverse, inner), and 5Ј-GGTCGTTTCGATGAACTGG-3Ј (reverse, outer). This BglII-BalI PCR fragment was cloned into a modified Myc-tagged SHP-1 construct.. Antibodies (anti-VSVG, 1:2500; anti-SHP-1, 1:1000; 4G10, 1:3000) were diluted in PBS containing 0.05% Tween 20 and incubated with the membranes for 1 h at room temperature. Proteins were visualized using the enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom)
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