Conformational Changes Induced in the Protein Tyrosine Kinase p72^syk by Tyrosine Phosphorylation or by Binding of Phosphorylated Immunoreceptor Tyrosine-Based Activation Motif Peptides

Abstract

(43,47,58). This motif, called the immunoreceptor tyrosine-based activation motif (ITAM), is present in the ␤ and ␥ chains of FcεRI, in the subunit of the T-cell receptor complex, and in immunoglobulin ␤ (Ig␤) and Ig␣ of the B-cell receptor (10, 12). Chimeric proteins with the cytoplasmic domain of or FcεRI␥ linked to the extracellular and transmembrane domains of other proteins have been used for signal transduction studies (17,22,24,34,45). Aggregation of these chimeric molecules by antibodies to their extracellular domains results in cell activation (21, 44). After receptor aggregation, the Syk/ZAP-70 protein tyrosine kinases associate with these receptors and are critical for the downstream activation signals (6,10,12,13,19,31).The Syk/ZAP-70 family of protein tyrosine kinases has two tandem Src homology 2 (SH2) domains in the N-terminal half that interact with specific phosphorylated tyrosine residues in other proteins (6,14,28,30,37,55,57). It is postulated that aggregation of these ITAM-containing receptors results in their phosphorylation on the tyrosine residues in the ITAMs probably because of a Src family protein tyrosine kinase (23). The tyrosine-phosphorylated ITAM then becomes the binding site for the Syk/ZAP-70 family of molecules (32). Although there is some association of Syk/ZAP-70 in the absence of receptor stimulation, the predominant interaction is with receptors that are activated and therefore tyrosine phosphorylated (13). For example, in the RBL-2H3 rat mast cell line aggregation of FcεRI results in the activation of Lyn and in the tyrosine phosphorylation of the ␤ and ␥ subunits of the receptor. Because of SH2-mediated interactions, there is increased association of Lyn with FcεRI␤ and the recruitment of Syk by the tyrosine-phosphorylated ␥ subunit of the receptor (6, 28, 48).The Syk/ZAP-70 tyrosine kinases that are bound to the ITAMs are usually tyrosine phosphorylated and activated (13,14,16). In transient transfection experiments, the activation of Syk/ZAP-70 required the presence of the Src family kinase (23). It is therefore postulated that the Src family kinase may tyrosine phosphorylate the Syk/ZAP-70 and induce activation (23). Here we present evidence that the binding of Syk to a diphosphorylated ITAM peptide results in a conformational change and in an increase in kinase activity. This change is likely involved in the downstream propagation of tyrosine phosphorylation signals (4, 5, 7). MATERIALS AND METHODSMaterials. Protein A-agarose, aprotinin, Triton X-100, and ATP were obtained from Sigma (St. Louis, Mo.). The materials for electrophoresis were purchased from NOVEX (San Diego, Calif.), polyvinylidene difluoride transfer membrane was purchased from Millipore (Bedford, Mass.), and the sources of other materials not indicated in this section were as described previously (6).Antibodies. Two peptides based on the rat p72 syk deduced amino acid sequence were synthesized with an additional Cys residue at either the carboxy or the N terminus to allow coupling to proteins. Antibod...

Cloning and characterization of APS, an adaptor molecule containing PH and SH2 domains that is tyrosine phosphorylated upon B-cell receptor stimulation

“…Iga chain may physically associate with Lyn or Fyn (Clark et al, 1992). After tyrosine-phosphorylation by these Src-family PTKs, the ITAMs recruit the Syk and ZAP70 PTKs to BCR and TCR complexes respectively (Wange et al, 1993;Iwashima et al, 1994;Kimura et al, 1996). Activated PTKs phosphorylate numerous cellular proteins, including phospholipase C (PLC)g1, Vav proto-oncogene product and adaptor proteins such as Shc and Crk (Carter et al, 1991;Bustelo and Barbacid, 1992;Panchamoorthy et al, 1996;Tezuka et al, 1996).…”

Tyrosines in the Carboxyl Terminus Regulate Syk Kinase Activity and Function

“…These phosphorylations create a docking site for binding by the two SH2 domains of Syk (16 -18). This binding of Syk to the phosphorylated ITAM results in a conformational change of Syk, with an increase in its enzymatic activity (19,20). Activation of Syk leads to downstream propagation of signaling with tyrosine phosphorylation of linker for activation of T cells (LAT), Vav, phospholipase C␥, and SH2 domain-containing leukocyte protein of 76 kDa (SLP-76).…”

Negative Signaling in Fc Receptor Complexes

“…In any case, phopshorylated ITAMs provide docking sites that mediate the recruitment of SH2 domain-containing molecules, among which is the two-SH2 domaincontaining protein tyrosine kinase Syk (Benhamou et al, 1993). Once recruited, Syk is tyrosyl-phosphorylated by src kinases and it further auto-phosphorylates (Kimura et al, 1996). This activates its catalytic activity.…”

“…As discussed above, molecules that contain two SH2 domains require the cooperative binding of these two domains to two sequences containing phosphorylated tyrosines in order to be recruited in vivo. The recruitment of ZAP-70 and Syk (Bu et al, 1995;Kurosaki et al, 1995), or SHP-1 (Lesourne et al, 2001), required the conservation of their two SH2 domains and the conservation of the two tyrosines of ITAMs in immunoreceptors (Kimura et al, 1996) or of the two ITIMs in KIRs (Bruhns et al, 1999;Burshtyn et al, 1999) respectively. Moreover, molecules that contain a single SH2 domain were found to require the cooperation of other SH2 domain-containing molecules in order to be recruited (Yamasaki et al, 2003).…”