Müller Spike Murphy, Nelson Nina, Jücker Manfred
Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
Biomolecules. 2025 Jan 10;15(1):105. doi: 10.3390/biom15010105.
The Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) is a multidomain protein consisting of two protein-protein interaction domains, the Src homology 2 (SH2) domain, and the proline-rich region (PRR), as well as three phosphoinositide-binding domains, the pleckstrin homology-like (PHL) domain, the 5-phosphatase (5PPase) domain, and the C2 domain. SHIP1 is commonly known for its involvement in the regulation of the PI3K/AKT signaling pathway by dephosphorylation of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P) at the D5 position of the inositol ring. However, the functional role of each domain of SHIP1 for the regulation of its enzymatic activity is not well understood. To determine the contribution of the individual domains to catalytic activity, the full-length protein was compared with truncated constructs lacking one or more domain(s), regarding the substrate turnover (k) and catalytic efficiency (k/K) towards ci8-PtdIns(3,4,5)P. With this approach, it was possible to verify the allosteric activation of SHIP1 mediated by the C2 domain as described previously, while the PHL domain seemed instead to have a negative effect regarding catalytic efficiency. The full-length SHIP1 clearly displayed the highest turnover and the second-highest catalytic efficiency, showing the role of the SH2 domain and PRR not only in protein-protein interactions but also in catalysis. The SH2 domain increased substrate turnover but negatively affected catalytic efficiency. The linker between the SH2 and the PHL domains decreased the turnover number but positively influenced the catalytic efficiency. The PRR increased both the substrate turnover and the protein's catalytic efficiency. The regression analysis of the Michaelis-Menten graph revealed SHIP1 to be an allosteric enzyme, with the PRR and the linker being the most involved domains in that regard. In summary, our data indicate a complex regulation of the enzymatic activity of SHIP1 by its individual domains. While the C2 domain and PRR at the carboxy-terminus have a positive effect on enzymatic activity, the SH2 and PHL domain at the amino-terminus inhibit catalytic efficiency.
含Src同源2结构域的肌醇5 -磷酸酶1（SHIP1）是一种多结构域蛋白，由两个蛋白质 - 蛋白质相互作用结构域，即Src同源2（SH2）结构域和富含脯氨酸区域（PRR），以及三个磷酸肌醇结合结构域，即类普列克底物蛋白同源（PHL）结构域、5 - 磷酸酶（5PPase）结构域和C2结构域组成。SHIP1因其通过使磷脂酰肌醇 - 3,4,5 - 三磷酸（PtdIns(3,4,5)P）在肌醇环的D5位置去磷酸化而参与PI3K/AKT信号通路的调节而广为人知。然而，SHIP1的每个结构域对其酶活性调节的功能作用尚不清楚。为了确定各个结构域对催化活性的贡献，将全长蛋白与缺失一个或多个结构域的截短构建体进行比较，比较它们对ci8 - PtdIns(3,4,5)P的底物周转率（k）和催化效率（k/K）。通过这种方法，可以验证如先前所述的由C2结构域介导的SHIP1的变构激活，而PHL结构域在催化效率方面似乎具有负面影响。全长SHIP1明显显示出最高的周转率和第二高的催化效率，表明SH2结构域和PRR不仅在蛋白质 - 蛋白质相互作用中起作用，而且在催化中也起作用。SH2结构域增加了底物周转率，但对催化效率有负面影响。SH2和PHL结构域之间的连接子降低了周转数，但对催化效率有正向影响。PRR增加了底物周转率和蛋白质的催化效率。米氏图的回归分析表明SHIP1是一种别构酶，在这方面PRR和连接子是最相关的结构域。总之，我们的数据表明SHIP1的酶活性受到其各个结构域的复杂调节。虽然羧基末端的C2结构域和PRR对酶活性有正向作用，但氨基末端的SH2和PHL结构域抑制催化效率。
