Sondhi D, Cole P A
Laboratory of Bioorganic Chemistry, The Rockefeller University, New York 10021, USA.
Biochemistry. 1999 Aug 24;38(34):11147-55. doi: 10.1021/bi990827+.
Csk (C-terminal Src kinase) is a protein tyrosine kinase that phosphorylates Src family member C-terminal tails, resulting in downregulation of Src family members. It is composed of three principal domains: an SH3 (Src homology 3) domain, an SH2 (Src homology 2) domain, and a catalytic domain. The impact of the noncatalytic domains on kinase catalysis was investigated. The Csk catalytic domain was expressed in Escherichia coli as a recombinant glutathione S-transferase-fusion protein and demonstrated to have 100-fold reduced catalytic efficiency. Production of the catalytic domain by proteolysis of full-length Csk afforded a similar rate reduction. This suggested that the reduction in catalytic efficiency of the recombinant catalytic domain was intrinsic to the sequence and not an artifact related to faulty expression. This rate reduction was similar for peptide and protein substrates and was due almost entirely to a reduced k(cat) rather than to effects on substrate K(m)s. Viscosity experiments on the catalytic fragment kinase reaction demonstrated that the chemical (phosphoryl transfer) step had a reduced rate. While the Csk SH2 domain had no intermolecular effect on the kinase activity of the Csk catalytic domain, the SH3 domain and SH3-SH2 fragment led to a partial rescue (4-5-fold) of the lost kinase activity. This rescue was not achieved with two other SH3 domains (lymphoid cell kinase, Abelson kinase). The extrapolated K(d) of interaction for the Csk catalytic domain with the Csk SH3 domain was 2.2 microM and that of the Csk catalytic domain with the Csk SH3-SH2 fragment was 8.8 microM. Taken together, these findings suggest that there is likely an intramolecular interaction between the catalytic and SH3 domains in full-length Csk that is important for efficient catalysis. By employing a Csk SH3 specific type II polyproline helix peptide and carrying out site-directed mutagenesis, it was established that the SH3 surface that interacts with the catalytic domain was distinct from the surface that binds type II polyproline helix peptides. This finding suggests a novel mode of protein-protein interaction for an SH3 domain. The implications for Csk substrate selectivity, regulation, and function are discussed.
Csk（C末端Src激酶）是一种蛋白质酪氨酸激酶，可使Src家族成员的C末端尾巴磷酸化，从而导致Src家族成员的下调。它由三个主要结构域组成：一个SH3（Src同源结构域3）结构域、一个SH2（Src同源结构域2）结构域和一个催化结构域。研究了非催化结构域对激酶催化的影响。Csk催化结构域在大肠杆菌中作为重组谷胱甘肽S-转移酶融合蛋白表达，其催化效率降低了100倍。通过全长Csk的蛋白水解产生催化结构域也导致了类似的速率降低。这表明重组催化结构域催化效率的降低是序列固有的，而不是与错误表达相关的假象。这种速率降低对于肽和蛋白质底物是相似的，并且几乎完全是由于k(cat)降低而不是对底物K(m)的影响。对催化片段激酶反应的粘度实验表明，化学（磷酰转移）步骤的速率降低。虽然Csk SH2结构域对Csk催化结构域的激酶活性没有分子间影响，但SH3结构域和SH3-SH2片段导致部分恢复（4-5倍）丧失的激酶活性。另外两个SH3结构域（淋巴细胞激酶、阿贝尔森激酶）未实现这种恢复。Csk催化结构域与Csk SH3结构域相互作用的外推K(d)为2.2 microM，Csk催化结构域与Csk SH3-SH2片段相互作用的外推K(d)为8.8 microM。综上所述，这些发现表明全长Csk的催化结构域和SH3结构域之间可能存在分子内相互作用，这对有效催化很重要。通过使用Csk SH3特异性II型多聚脯氨酸螺旋肽并进行定点诱变，确定与催化结构域相互作用的SH3表面与结合II型多聚脯氨酸螺旋肽的表面不同。这一发现提示了SH3结构域一种新的蛋白质-蛋白质相互作用模式。讨论了其对Csk底物选择性、调节和功能的影响。
