Vallano M L, Goldenring J R, Buckholz T M, Larson R E, DeLorenzo R J
Proc Natl Acad Sci U S A. 1985 May;82(10):3202-6. doi: 10.1073/pnas.82.10.3202.
Both cAMP- and calmodulin-dependent kinases are proposed regulators of microtubule function by means of their ability to phosphorylate microtubule-associated protein 2(MAP 2). A cAMP-dependent kinase/MAP 2 complex is endogenous to microtubules. In this report, we demonstrate that an endogenous calmodulin-dependent kinase that phosphorylates MAP 2 as a major substrate is also present in microtubules prepared under conditions that preserve kinase activity. This enzyme is identical to a calmodulin-dependent kinase purified previously from rat brain cytosol. A fraction containing calmodulin-dependent kinase and MAP 2 was separated from the cAMP-dependent kinase/MAP 2 complex by gel filtration chromatography of microtubule protein in high ionic strength buffer. All of the recovered calmodulin-dependent kinase activity in microtubules eluted in a single protein peak. The specific activity of the enzyme for MAP 2 was enriched 31-fold in this fraction compared to cytosol. Two-dimensional tryptic phosphopeptide mapping revealed that the endogenous cAMP- and calmodulin-dependent kinases phosphorylated distinct sites on MAP 2. These data demonstrate that both kinases are present in microtubule preparations and that they may differentially regulate MAP 2 function by phosphorylating separate sites on MAP 2.
环磷酸腺苷（cAMP）依赖性激酶和钙调蛋白依赖性激酶都被认为是微管功能的调节因子，因为它们具有磷酸化微管相关蛋白2（MAP 2）的能力。一种cAMP依赖性激酶/MAP 2复合物是微管内源性的。在本报告中，我们证明，在保留激酶活性的条件下制备的微管中也存在一种内源性钙调蛋白依赖性激酶，该激酶以MAP 2作为主要底物进行磷酸化。这种酶与先前从大鼠脑细胞质中纯化的钙调蛋白依赖性激酶相同。通过在高离子强度缓冲液中对微管蛋白进行凝胶过滤色谱，从cAMP依赖性激酶/MAP 2复合物中分离出含有钙调蛋白依赖性激酶和MAP 2的组分。微管中所有回收的钙调蛋白依赖性激酶活性都在一个单一的蛋白峰中洗脱出来。与细胞质相比，该组分中该酶对MAP 2的比活性提高了31倍。二维胰蛋白酶磷酸肽图谱显示，内源性cAMP依赖性激酶和钙调蛋白依赖性激酶在MAP 2上磷酸化不同的位点。这些数据表明，两种激酶都存在于微管制剂中，并且它们可能通过磷酸化MAP 2上不同的位点来差异调节MAP 2的功能。
