Rider M H, Vandamme J, Lebeau E, Vertommen D, Vidal H, Rousseau G G, Vandekerckhove J, Hue L
Hormone and Metabolic Research Unit, International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
Biochem J. 1992 Jul 15;285 ( Pt 2)(Pt 2):405-11. doi: 10.1042/bj2850405.
Purified bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) showed two bands with subunit M(r) of 58,000 and 54,000 when analysed by SDS/PAGE. Both the 58,000- and 54,000-M(r) forms were phosphorylated by cyclic AMP-dependent protein kinase (PKA) and by protein kinase C (PKC) in vitro. Phosphorylation by PKA decreased the apparent Km of PFK-2 for one of its substrates, fructose 6-phosphate, while phosphorylation by PKC did not correlate with any change in PFK-2 activity. The differences between the 58,000- and 54,000-M(r) forms were studied by electroblotting, peptide mapping and microsequencing. Residues 451-510, which correspond to exon 15 in the rat and contain phosphorylation sites for PKA (Ser-466) and PKC (Thr-475), were absent from the 54,000-M(r) form. Peptide mapping after phosphorylation by [gamma-32P]MgATP and PKC showed a phosphorylated peptide containing Thr-475, which was present in the 58,000-M(r) form but not in the 54,000-M(r) form. The fact that the latter form was phosphorylated by PKC and PKA suggests that other phosphorylation sites for PKA and PKC are located outside the region encoded by exon 15. Finally, analysis of RNA from bovine heart showed that the tissue contains two PFK-2/FBPase-2 mRNAs, only one of which was recognized by a probe specific to the region coding for Ser-466 and Thr-475. Taken together, these findings demonstrate that the 58,000- and 54,000-M(r) forms of bovine heart PFK-2/FBPase-2 result from alternative splicing of the same primary transcript.
经十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）分析，纯化的牛心6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶（PFK-2/FBPase-2）呈现出两条亚基分子量分别为58,000和54,000的条带。在体外，分子量为58,000和54,000的这两种形式均能被环磷酸腺苷依赖性蛋白激酶（PKA）和蛋白激酶C（PKC）磷酸化。PKA介导的磷酸化降低了PFK-2对其底物之一果糖6-磷酸的表观米氏常数（Km），而PKC介导的磷酸化与PFK-2活性的任何变化均无关联。通过电转印、肽图谱分析和微量测序研究了分子量为58,000和54,000的这两种形式之间的差异。分子量为54,000的形式缺少对应于大鼠外显子15且包含PKA磷酸化位点（丝氨酸-466）和PKC磷酸化位点（苏氨酸-475）的第451 - 510位氨基酸残基。用[γ-32P]MgATP和PKC进行磷酸化后的肽图谱分析显示，存在一条含有苏氨酸-475的磷酸化肽段，该肽段存在于分子量为58,000的形式中，而不存在于分子量为54,000的形式中。分子量为54,000的形式能被PKC和PKA磷酸化这一事实表明，PKA和PKC的其他磷酸化位点位于外显子15编码区域之外。最后，对牛心RNA的分析表明，该组织含有两种PFK-2/FBPase-2 mRNA，其中只有一种能被针对编码丝氨酸-466和苏氨酸-475区域的探针识别。综上所述，这些发现表明，牛心PFK-2/FBPase-2的分子量为58,000和54,000的两种形式是同一初级转录本选择性剪接的结果。
