Identification of new JNK substrate using ATP pocket mutant JNK and a corresponding ATP analogue

Modification of the ATP pocket on protein kinases allows selective use of a n ATP analogue that exhibits high affinity for the altered kinases. Using t his approach, we altered the ATP-binding site on JNK and identified N-6-(2- phenythyl)-ATP, a modified form of ATP that exhibits high specificity and a ffinity for the modified, but not the wild type form, of JNK. Using modifie d JNK and its ATP analogue enables the detection of novel JNK substrates. A mong substrates identified using this approach is heterogeneous nuclear rib onucleoprotein K, which is involved in transcription and post-transcription al mRNA metabolism. The newly identified substrate can be phosphorylated by JNK on amino acids 216 and 353, which contribute to heterogeneous nuclear ribonucleoprotein K mediated transcriptional activities.