BIT (a brain immunoglobulin-like molecule with tyrosine-based activation motifs) is a brain-specific membrane protein which has two cytoplasmic TAMs (tyrosine-based activation motifs). Using the Far Western blotting technique, we detected association of a 70-kDa protein with the tyrosine-phosphorylated TAMs of BIT. A mouse brain cDNA library in gt11 was screened for this association, and two positive clones encoding tyrosine phosphatase SH-PTP2 were isolated. SH-PTP2 has two SH2 domains and is believed to function as a positive mediator in receptor tyrosine kinase signaling. SH-PTP2 and BIT were coimmunoprecipitated from phosphorylated rat brain lysate, and BIT was a major tyrosine-phosphorylated protein associated with SH-PTP2 in this lysate. This interaction was also observed in Jurkat T cells transfected with BIT cDNA depending on tyrosine phosphorylation of BIT. Bisphosphotyrosyl peptides corresponding to BIT-TAMs stimulated SH-PTP2 activity 33-35-fold in vitro, indicating that two SH2 domains of SH-PTP2 simultaneously interact with two phosphotyrosines of BIT-TAM. Our findings suggest that the tyrosine phosphorylation of BIT results in stimulation of the signal transduction pathway promoted by SH-PTP2 and that BIT is probably a major receptor molecule in the brain located just upstream of SH-PTP2.Protein tyrosine phosphorylation plays an important role in signal transduction and regulates a wide range of cellular processes. Protein-tyrosine kinases and protein-tyrosine phosphatases are highly expressed in the central nervous system, consistent with the importance of tyrosine phosphorylation in neural function (1).BIT 1 (a brain immunoglobulin-like molecule with tyrosinebased activation motifs) is a novel immune antigen receptorlike molecule of the brain.2 This molecule is composed of an antigen receptor-like extracellular domain, a transmembrane domain, and a cytoplasmic region containing two variants of TAM (tyrosine-based activation motif) that was recently designated ITAM (immunoreceptor TAM). This cytoplasmic motif contains two tyrosine phosphorylation sites. TAM was originally described in the immune system where it plays a crucial role in the activation responses of B and T cells (2-5). BIT is one of major substrates of protein-tyrosine kinase(s) in crude brain suspensions 2 and is widely distributed in the brain in synapse-rich regions and in some nerve fibers.3 These findings suggest that the tyrosine phosphorylation of TAMs in BIT may be involved in neural signal transduction. Recent studies in the immune system have demonstrated that the oligomerization of TAMs allows the phosphorylation of two tyrosine residues found in this motif and these phosphotyrosine residues act as a bidentate docking site for the paired Src homology 2 (SH2) domains present in the cytoplasmic tyrosine kinases, Syk and ZAP-70, believed to be indispensable for initiation of the signaling cascade (6 -8). From these investigations, we predicted that TAMs of BIT may recruit tyrosine kinases containing paired SH2 domains to the ...
Hyperphosphorylated tau protein is known to be a major component of the paired helical filaments (PHFs) that accumulate in the brain of Alzheimer's patients. The kinase that phosphorylated Ser-208 and Ser-210 in PHF-tau had remained unknown. We used anti-pS208 and anti-pS210 antibodies and Western blots to confirm that the tau-tubulin kinase (TTK) phosphorylates tau at Ser-208 and at Ser-210. Using partial amino acid sequences of purified bovine brain TTK, a mouse cDNA of TTK was isolated and the sequence was determined. Its 963 bp coding region is composed of 320 amino acids and encodes a 36 kDa protein indistinguishable in size from authentic bovine brain TTK. Our immunoblot analysis demonstrated that TTK is ubiquitously distributed in the rat tissues, and that it is developmentally regulated in the rat brain. ß
Conotoxins are multiple disulfide-bonded peptides isolated from marine cone snail venom. These toxins have been classified into several families based on their disulfide pattern and biological properties. Here, we report a new family of Conus peptides, which have a novel cysteine motif. Three peptides of this family (CMrVIA, CMrVIB, and CMrX) have been purified from Conus marmoreus venom, and their structures have been determined. Their amino acid sequences are VCCGYK-LCHOC (CMrVIA), NGVCCGYKLCHOC (CMrVIB), and GICCGVSFCYOC (CMrX), where O represents 4-transhydroxyproline. Two of these peptides (CMrVIA and CMrX) have been chemically synthesized. Using a selective protection and deprotection strategy during disulfide bond formation, peptides with both feasible cysteine-pairing combinations were generated. The disulfide pattern (C 1 -C 4 , C 2 -C 3 ) in native toxins was identified by their co-elution with the synthetic disulfideisomeric peptides on reverse-phase high pressure liquid chromatography. Although cysteine residues were found in comparable positions with those of ␣-conotoxins, these toxins exhibited a distinctly different disulfide bonding pattern; we have named this new family " -conotoxins." CMrVIA and CMrX induced different biological effects when injected intra-cerebroventricularly in mice; CMrVIA induces seizures, whereas CMrX induces flaccid paralysis. The synthetic peptide with -conotoxin folding is about 1150-fold more potent in inducing seizures than the mispaired isomer with ␣-conotoxin folding. Thus it appears that the unique disulfide pattern, and hence the "ribbon" conformation, in -conotoxins is important for their biological activity.Conotoxins are biologically active peptide toxins isolated from venomous marine cone snails. They are typically small disulfide-rich peptides containing 11-30 amino acid residues. Conotoxins can be classified into several families based on the number and pattern of disulfide bonds and biological activities (1, 2). Members of a single family of conotoxins share similar protein folding but in some cases exhibit different biological activities (3-5). These differences in biological activities are due to their ability to bind with specific ion channels or receptors (6). Some of these conotoxins are used as tools in investigating receptor structure and function (2) and ion channel geometry (7). The structurally constrained scaffolds of conotoxins are utilized as a template for protein engineering to create chimeric proteins (8, 9).Conotoxins, like toxins from other venoms, evolve rapidly by positive Darwinian selection (10). The specificity of conotoxins is due to their disulfide bonding framework and specific amino acids in inter-cysteine loops. The high density of disulfides in conotoxins plays a vital role in their stability and imposes a distinct protein folding with a specific orientation of hypervariable loop regions. The diversity in conotoxins is also achieved through disulfide pairings (see "Discussion").The conotoxins with two disulfide bonds can have th...
During purification oftau protein kinase I and II from the bovine brain extract, a new tau protein kinase was detected and purified with phosphocellulose, gel filtration, S-Sepharose and AF-Heparin column chromatography. The molecular mass of the enzyme was determined to be 32 kDa by gel filtration and activity staining on SDS-PAGE. The enzyme is a Ser/Thr protein kinase phosphorylating tau, l~-tubulin, MAP2 and ~-casein. Employing many synthetic peptides, the recognition site of this enzyme appears to be -SR-. The enzyme requires no second messenger and is inhibited with high concentration of heparin, but not by inhibitors of CKI. These results indicate that this enzyme, tau-tubulin kinase is novel and distinct from TPKI, II and CKI, II.
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