CONFORMATION-DEPENDENT PHOSPHORYLATION OF P53

Phosphorylation of the p53 tumor suppressor protein is known to modula te its functions, Using bacterially produced glutathione S-transferase (GST)-p53 fusion protein and baculovirus-expressed histidine-tagged p 53 (Hi,p53), we have determined human p53 phosphorylation by purified forms of jun-N-kinase (JNK), protein kinase A (PKA), and beta subunit of casein kinase II (CKII beta) as well as by kinases present in whole cell extracts (WCEs), We demonstrate that PKA is potent p53 kinase, a lbeit, in a conformation- and concentration-dependent manner, as concl uded by comparing full-length with truncated forms of p53, We further demonstrate JNK interaction with GST;p53 and the ability of JNK to pho sphorylate truncated forms of GST-p53 or full-length (His)p53. Depende nce of phosphorylation on conformation of p53 is further supported by the finding that the wild-type form of p53 (p53(wt)) undergoes better phosphorylation by CKII beta and by WCE kinases than mutant forms of p 53 at amino acid 249 (p53(249)) or 273 (p53(273)), Moreover, shifting the kinase reaction's temperature from 37 degrees C to 18 degrees C re duces the phosphorylation of mutant p53 to a greater extent than of p5 3(wt)., Comparing truncated forms of p53 revealed that the ability of CKII beta, PKA, or WCE kinases to phosphorylate p53 requires amino aci ds 97-155 within the DNA-binding domain region, Among three 20-aa pept ides spanning this region we have identified residues 97-117 that incr ease p53 phosphorylation by CKII beta while inhibiting p53 phosphoryla tion by PKA or WCE kinases, The importance of this region is further s upported by computer modeling studies, which demonstrated that mutant p53(249) exhibits significant changes to the conformation of p53 withi n amino acids 97-117, In summary, phosphorylation-related analysis of different p53 forms in vitro indicates that conformation of p53 is a k ey determinant in its availability as a substrate for different kinase s, as for the phosphorylation pattern generated by the same kinase.