Purification and characterization of a protein‐tyrosine kinase p72^syk from porcine spleen

Abstract

We have succeeded in purifying p7Tyk, a non-receptor-type protein-tyrosine kinase carrying high susceptibility to proteolysis [Taniguchi, T., Kobayashi, T., Kondo, J., Takahashi, K., Nakamura, H., Suzuki, J., Nagai, K., Yamada, T., Nakamura, S. and Yamamura, H. (1991) J. BioE. Chenz. 266, 15 790 -157961 from porcine spleen. The purification procedure involves a sequential column chromatography, following extraction with 0.5 M NaCl from spleen homogenate, on phosphocellulose, Sephacryl S-200, heparin-Sepharose CL-6B, Mono Q and Mono S. SDSPAGE of the final purified sample revealed a 72-kDa protein band with about 95% purity and immunodepletion analysis showed immunological cross-reactivity with anti-p7Tyk antibody which does not recognize ZAP-70. It was purified approximately 3000-fold with an overall yield of 0.54% according to [ValSIangioten-sin I1 phosphorylation activity and the specific activity of the final sample (30 nmol phosphate .min-' . mg protein-') was relatively lower than that of the 40-kDa kinase, a catalytic fragment of p72yk which lacks two src homology regions 2 domains.The ~7 2 "~ had an autophosphorylation activity that was performed by intramolecular catalysis accompanied by a phosphate exchange reaction, and could efficiently phosphorylate tubulin, myelin basic protein and H2B histone. Employing [ValSIangiotensin I1 as a substrate, the apparent K,,, value for the peptide was 0.91 mM and that for ATP was 0.48 pM. Mn2+, Mg2+ and Co2+ were effective divalent cations and optimum pH was around 8.0-8.5 for the expression of the activity.These results suggest that the purified p7Tyk may exist as a less active form compared with the 40-kDa kinase and that the part of ~7 2 "~ containing two src homology region 2 domains may participate in the regulation of its activity though the enzymic character is quite similar to that of the 40-kDa kinase.A number of protein-tyrosine kinases (PTK) have been identified so far and can be broadly categorized into two families according to the structure : a transmembrane receptor family and a non-receptor family. The receptor family is believed to initiate the action of the ligand, such as epidermal growth factor, platelet-derived growth factor, insulin and so forth, by binding to it and raising the intrinsic kinase activity. In the case of the non-receptor family, more than ten members have been reported so far, including the syk gene product. However, their function, such as an activation mechanism as well as their position in a signal transduction pathway, is still unclear compared to those of the receptor family [l-51.Previously, we purified a 40-kDa PTK from cytosolic fractions of porcine spleen [6]. Consecutively, the molecular cloning of a gene syk (designated after spleen tyrosine kinase) revealed that it encodes a 72-kDa PTK (p7Tyk) belonging to the non-receptor family and that the 40-kDa kinase was its catalytic fragment generated by proteolysis in the purification procedure. The deduced amino-acid sequence of p72yk has a distinct character from other memb...

Purification and Characterization of Human ZAP-70 Protein-tyrosine Kinase from a Baculovirus Expression System

“…This value is in good agreement with the K m for ATP reported for other PTKs such as p60 src (ϳ2.2 (51), ϳ4.0 (52), and ϳ80.0 M (53)) and Csk (ϳ8 (54) and ϳ11.5 M (55)). The K m and V max values for cfb3 phosphorylation by ZAP-70 were ϳ2.5 M and ϳ7.4 nmol⅐min Ϫ1 ⅐mg Ϫ1 , respectively, which are consistent with what has been previously observed for the closely related Syk PTK (32,40).…”

“…This system has been used to express a number of recombinant PTKs that are involved in T cell activation including Lck (30,31) and Syk (32). We used this system for expression and purification of the human ZAP-70 PTK.…”

SH2 domain–mediated targeting, but not localization, of Syk in the plasma membrane is critical for FcεRI signaling

“…50 This suggests that there are intramolecular mechanisms to regulate Syk enzyme activity. Similarly, intramolecular interactions regulate the catalytic activity of the Src kinases.…”

The Spleen Protein‐Tyrosine Kinase TPK‐IIB is Highly Similar to the Catalytic Domain of p7Psyk

“…To obtain further evidence for a relationship between the 38-kDa catalytic component of TPK-IIB and p7Tyk, the latter was purified from rat spleen according to Yang et al (1994) (see the Materials and Methods section) and its biochemical properties were compared to those of TPK-IIB. Such a comparison is especially meaningful since the specificity of TPK-IIB shows some unique features.…”