Bannister A J, Brown H J, Sutherland J A, Kouzarides T
Wellcome/CRC Institute, Cambridge, UK.
Nucleic Acids Res. 1994 Dec 11;22(24):5173-6. doi: 10.1093/nar/22.24.5173.
The c-Fos and c-Jun proteins bind an AP1 site and activate transcription synergistically. These two proteins have a common activation domain which has two co-operating motifs, HOB1 and HOB2. The HOB1 motif of c-Jun includes S73 which is required for Ha-Ras-induced super-activation and phosphorylation by MAP kinase-like enzymes. Since c-Fos HOB1 has a conserved Thr residue (T232) analogous to c-Jun S73 we have proposed that c-Fos HOB1 will be regulated in the same way as c-Jun HOB1. Here we show that the HOB1-containing activation domain of c-Fos is stimulated by Ha-Ras in vivo and phosphorylated by a MAP kinase family member in vitro and that mutating T232 to Ala abolishes both functions. Collectively these results suggest that phosphorylation of the HOB1 motif increases its activation capacity. To provide direct evidence for this we change the context of c-Fos T232 to a PKA recognition site, and show that HOB1 activity is now stimulated by the catalytic subunit of PKA. This 'PKA specificity' experiment represents a novel and powerful way to analyse phosphorylation events involved in a variety of biological functions.
c-Fos和c-Jun蛋白结合一个AP1位点并协同激活转录。这两种蛋白有一个共同的激活结构域，该结构域有两个协同基序，即HOB1和HOB2。c-Jun的HOB1基序包括S73，它是Ha-Ras诱导的超激活以及被MAP激酶样酶磷酸化所必需的。由于c-Fos的HOB1有一个与c-Jun的S73类似的保守苏氨酸残基（T232），我们提出c-Fos的HOB1将以与c-Jun的HOB1相同的方式受到调控。在此我们表明，c-Fos含HOB1的激活结构域在体内受Ha-Ras刺激，在体外被一个MAP激酶家族成员磷酸化，并且将T232突变为丙氨酸会消除这两种功能。总体而言，这些结果表明HOB1基序的磷酸化增加了其激活能力。为了对此提供直接证据，我们将c-Fos T232的环境改变为PKA识别位点，并表明HOB1活性现在受到PKA催化亚基的刺激。这个“PKA特异性”实验代表了一种分析参与多种生物学功能的磷酸化事件的新颖且强大的方法。
