Prickett Todd D, Brautigan David L
Center for Cell Signaling and Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
J Biol Chem. 2006 Oct 13;281(41):30503-11. doi: 10.1074/jbc.M601054200. Epub 2006 Aug 8.
The protein Ser/Thr phosphatase family contains three enzymes called PP2A, PP4, and PP6 with separate biological functions inferred from genetics of the yeast homologues Pph21/22, Pph3, and Sit4. These catalytic subunits associate with a common subunit called alpha4 (related to yeast Tap42). Here, we characterized recombinant PP6 and PP2A catalytic monomers and alpha4.phosphatase heterodimers. Monomeric PP6 and PP2A showed identical kinetics using either p-nitrophenyl phosphate (pNPP) or 32P-myelin basic protein (MBP) as substrates, with matching Km and Vmax values. Using pNPP as substrate, PP6 and PP2A gave the same IC50 with active site inhibitors okadaic acid, microcystin-LR, calyculin A, and cantharidin. However, with MBP as substrate, PP6 was inhibited at 5-fold lower concentrations of toxins relative to PP2A, suggesting PP6 might be a preferred in vivo target of toxins. Heterodimeric alpha4.PP6 and alpha4.PP2A were starkly different. With MBP as substrate the alpha4.PP2A heterodimer had a 100-fold higher Vmax than alpha4.PP6, and neither heterodimer was active with pNPP. Thus, these phosphatases are distinguished by their different responses to allosteric binding of the common regulatory subunit alpha4. Transient expression of alpha4 differentially increased or decreased phosphorylation of endogenous phosphoproteins, consistent with opposing effects on PP2A and PP6.
丝氨酸/苏氨酸磷酸酶家族包含三种酶，分别称为PP2A、PP4和PP6，其各自的生物学功能可从酵母同源物Pph21/22、Pph3和Sit4的遗传学研究中推断得出。这些催化亚基与一个名为alpha4（与酵母Tap42相关）的共同亚基结合。在此，我们对重组PP6和PP2A催化单体以及alpha4 - 磷酸酶异二聚体进行了表征。单体PP6和PP2A使用对硝基苯磷酸酯（pNPP）或32P - 髓鞘碱性蛋白（MBP）作为底物时表现出相同的动力学，具有匹配的Km和Vmax值。以pNPP为底物时，PP6和PP2A对活性位点抑制剂冈田酸、微囊藻毒素 - LR、花萼海绵诱癌素A和斑蝥素具有相同的IC50。然而，以MBP为底物时，相对于PP2A，PP6在低5倍浓度的毒素作用下就受到抑制，这表明PP6可能是毒素在体内的首选作用靶点。异二聚体alpha4 - PP6和alpha4 - PP2A则明显不同。以MBP为底物时，alpha4 - PP2A异二聚体的Vmax比alpha4 - PP6高100倍，并且两种异二聚体对pNPP均无活性。因此，这些磷酸酶的区别在于它们对共同调节亚基alpha 的变构结合有不同的反应。alpha4的瞬时表达差异性地增加或减少了内源性磷酸化蛋白的磷酸化，这与对PP2A和PP6的相反作用一致。
