Feng J F, Readon M, Yadav S P, Im M J
Department of Molecular Cardiology, The Molecular Biotechnology Core, The Lerner Research Institute, The Cleveland Clinic Foundation, Ohio 44195, USA.
Biochemistry. 1999 Aug 17;38(33):10743-9. doi: 10.1021/bi9905009.
Enzyme regulation is an important mechanism for controlling cell proliferation and differentiation in response to extracellular signaling molecules. We have previously reported that a approximately 50 kDa protein (termed Gbetah) consistently copurified with Galphah (transglutaminase II, TGII) and that Gbetah down-regulates the GTPase function of TGII by associating with GDP-bound TGII [Baek et al. (1996) Biochemistry 35, 2651-2657]. In this study, we examined the identity of Gbetah by partial amino acid sequencing and immunological characterizations. The results strongly suggest that Gbetah is a protein known as calreticulin (CRT). When the regulatory role of CRT in the GTPase activity of TGII was examined, CRT inhibited GTP (GTPgammaS) binding and hydrolysis in a concentration-dependent manner. Moreover, CRT interacted only with GDP-bound TGII. These results demonstrate that CRT down-regulates the GTPase activity of TGII by associating with GDP-bound TGII. Studies on the modulation of the TGase activity of TGII revealed that CRT also inhibited TGase activity. The inhibition showed the two characteristics depend on guanine nucleotides occupying the GTPase active site. The inhibition of the "empty" form of the GTPase active site increased the Ca2+ requirement without changing the Vmax. On the other hand, the inhibition of the GDP-bound form decreased Vmax, but did not alter the Ca2+ requirement. Moreover, the GTPgammaS-bound TGII was virtually resistant to Ca2+-mediated stimulation of the TGase activity, indicating that the GTP-bound TGII does not function as a TGase. We concluded that CRT is the regulatory protein of TGII that down-regulates both GTPase and TGase activities, opposing the activators of TGII function.
酶调节是一种重要机制，可响应细胞外信号分子来控制细胞增殖和分化。我们之前报道过，一种约50 kDa的蛋白质（称为Gbetah）始终与Galphah（转谷氨酰胺酶II，TGII）共纯化，并且Gbetah通过与结合GDP的TGII缔合来下调TGII的GTP酶功能[Baek等人（1996年）《生物化学》35卷，2651 - 2657页]。在本研究中，我们通过部分氨基酸测序和免疫学特征鉴定来研究Gbetah的身份。结果强烈表明Gbetah是一种名为钙网蛋白（CRT）的蛋白质。当研究CRT对TGII的GTP酶活性的调节作用时，CRT以浓度依赖的方式抑制GTP（GTPγS）结合和水解。此外，CRT仅与结合GDP的TGII相互作用。这些结果表明，CRT通过与结合GDP的TGII缔合来下调TGII的GTP酶活性。对TGII的转谷氨酰胺酶活性调节的研究表明，CRT也抑制转谷氨酰胺酶活性。该抑制表现出两个取决于占据GTP酶活性位点的鸟嘌呤核苷酸的特征。对GTP酶活性位点“空”形式的抑制增加了对Ca2 +的需求，而不改变Vmax。另一方面，对结合GDP形式的抑制降低了Vmax，但没有改变对Ca2 +的需求。此外，结合GTPγS的TGII实际上对Ca2 +介导的转谷氨酰胺酶活性刺激具有抗性，表明结合GTP的TGII不发挥转谷氨酰胺酶的功能。我们得出结论，CRT是TGII的调节蛋白，可下调GTP酶和转谷氨酰胺酶活性，与TGII功能的激活剂相反。
