| Then , by affinity chromatographies and in vitro kinase assays with fusion proteins between glutathione S transferase and the transactivator domain of Spi B , two kinases were identified on their ability to interact and phosphorylate this domain ; the MAP kinase ERK 1 and the stress activated protein kinase JNK 1 . ^^^ The Threonine 56 was defined as the ERK 1 phosphorylation site by using phosphoamino acid analyses and a Spi B mutant version with the substitution T 56 to A 56 . ^^^ Strikingly , ERK 1 failed to phosphorylate Spi 1 , in vitro , whereas JNK 1 , like CK 2 , phosphorylated Spi B and Spi 1 . ^^^ In addition , other purified Spi B kinase activities , unidentified as yet , display similar specificity than ERK 1 for Spi B versus Spi 1 . ^^^ Furthermore , the evident interaction of pRb protein with the transactivator domain of Spi B in an unphosphorylated state disappeared when this domain was first phosphorylated in vitro either by ERK 1 or by the purified Spi B kinase activities . ^^^ |