Durán Rosario, Villarino Andrea, Bellinzoni Marco, Wehenkel Annemarie, Fernandez Pablo, Boitel Brigitte, Cole Stewart T, Alzari Pedro M, Cerveñansky Carlos
Laboratorio de Bioquímica Analítica, Instituto de Investigaciones Biológicas Clemente Estable and Facultad de Ciencias, Montevideo, Uruguay.
Biochem Biophys Res Commun. 2005 Aug 5;333(3):858-67. doi: 10.1016/j.bbrc.2005.05.173.
The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to establish interaction networks involving signaling elements. Using different strategies to identify phosphorylated residues, we report here mass spectrometry studies of the entire intracellular regions of four 'receptor-like' protein kinases from Mycobacterium tuberculosis (PknB, PknD, PknE, and PknF), each consisting of an N-terminal kinase domain and a juxtamembrane region of varying length (26-100 residues). The enzymes were observed to incorporate different numbers of phosphates, from five in PknB up to 11 in PknD or PknE, and all detected sites were dephosphorylated by the cognate mycobacterial phosphatase PstP. Comparison of the phosphorylation patterns reveals two recurrent clusters of pThr/pSer residues, respectively, in their activation loops and juxtamembrane regions, which have a distinct effect on kinase activity. All studied kinases have at least two conserved phosphorylated residues in their activation loop and mutations of these residues in PknB significantly decreased the kinase activity, whereas deletion of the entire juxtamembrane regions in PknB and PknF had little effect on their activities. These results reinforce the hypothesis that mycobacterial kinase regulation includes a conserved activation loop mechanism, and suggest that phosphorylation sites in the juxtamembrane region might be involved in putative kinase-mediated signaling cascades.
蛋白质磷酸化位点的鉴定为研究信号转导通路以及建立涉及信号元件的相互作用网络提供了一个强大的工具。我们采用不同策略鉴定磷酸化残基，在此报告对结核分枝杆菌4种“受体样”蛋白激酶（PknB、PknD、PknE和PknF）整个细胞内区域的质谱研究，每种激酶均由一个N端激酶结构域和一个长度各异（26 - 100个残基）的近膜区域组成。观察到这些酶掺入的磷酸数量不同，PknB中有5个，PknD或PknE中多达11个，并且所有检测到的位点都被同源的分枝杆菌磷酸酶PstP去磷酸化。磷酸化模式的比较揭示，在它们的激活环和近膜区域分别有两个重复出现的pThr/pSer残基簇，这对激酶活性有不同的影响。所有研究的激酶在其激活环中至少有两个保守的磷酸化残基，PknB中这些残基的突变显著降低了激酶活性，而PknB和PknF中整个近膜区域的缺失对其活性影响不大。这些结果强化了分枝杆菌激酶调节包括保守的激活环机制这一假说，并表明近膜区域的磷酸化位点可能参与了假定的激酶介导的信号级联反应。
