PEPTIDE SUBSTRATES FOR ASSAY OF EXTRACELLULAR SIGNAL-REGULATED PROTEIN KINASE 1 AND 2 ACTIVITY

摘要

Several synthetic peptides modeled after Ser31 in tyrosine hydroxylase (“Ser31 peptides”) have been developed and evaluated as in vitro substrates for assaying the activity of extracellular signal-regulated protein kinase 1 and 2 (“ERK1/2”). The phosphorylation of the Ser31 peptides by activated, recombinant ERK2 was found to exhibit catalytic efficiencies (V_max/K_m) up to 4-fold higher than that of a synthetic myelin basic protein (MBP)-based peptide. Several synthetic peptides were tested using cellular extracts from PC 12 rat pheochromocytoma cells, both untreated cells and cells treated with nerve growth factor. Although the phosphorylation of the MBP peptide by extracts of PC12 cells was higher than that of the Ser31 peptide, the relative treatment-dependent increase was much greater for the Ser31 peptide and the pattern of ERK1/2 activation more closely mimicked the pattern seen with more complicated assays that initially isolated ERK1/2 from other kinases in the cellular extracts. This result suggested that the Ser31 peptide was a more specific substrate for the ERK1/2. Use of the new Ser31 peptide substrates will decrease the amount of peptide required to assay for ERK1/2 activity. In addition, the higher catalytic efficiencies associated with greater specificity for ERK1/2 will enable researchers to assay for activity of ERK1/2 in cellular extracts without prior immunoprecipitation.