cAMP-dependent protein kinase, but not the cGMP-dependent enzyme, rapidly phosphorylates delta-CREB, and a synthetic delta-CREB peptide.
Phosphorylation of the cAMP response element binding protein (CREB) by the catalytic subunit of cAMP-dependent protein kinase (cAK) has been implicated in the cAMP-dependent stimulation of gene transcription. delta-CREB, a spliced variant of CREB, and CREBtide (KRREILSRRPSYR), a synthetic peptide based on the phosphorylation sequence in delta-CREB, were tested as substrates of cAK. Phosphorylation of delta-CREB (0.17 microM) was stoichiometric within 30 s when using a concentration of cAK which approximated the intracellular level (0.2 microM). The rate of phosphorylation of delta-CREB was comparable to the rates of the best physiological substrates of cAK tested. The rate of CREBtide phosphorylation was at least as great as that of delta-CREB, indicating that the peptide retained the determinants of delta-CREB which were responsible for substrate efficacy. The apparent Km of CREBtide phosphorylation by cAK was 3.9 microM, which is 10-fold lower than that of kemptide (Km = 39 microM), the synthetic peptide substrate most often employed for cAK measurement. The Vmax values were 12.4 mumol/(min.mg) for CREBtide and 9.8 mumol/(min.mg) for kemptide. The apparent Km of CREBtide phosphorylation by cGMP-dependent protein kinase (cGK) was 2.9 microM and the Vmax value was 3.2 mumol/(min.mg). Both delta-CREB and CREBtide were phosphorylated at a much slower rate by cGK as compared with cAK, implying that the high cAK/cGK specificity exhibited by delta-CREB was retained by the peptide. Taken together, the results indicated that delta-CREB and CREBtide are among the best substrates tested for cAK and suggested that phosphorylation of CREB by this enzyme could occur in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Colbran
J L
JL
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232-0615.
Roach
P J
PJ
Fiol
C J
CJ
Dixon
J E
JE
Andrisani
O M
OM
Corbin
J D
JD
D000595:Amino Acid Sequence; D000242:Cyclic AMP / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D006152:Cyclic GMP / Q000502:physiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity
D017362:Cyclic AMP Response Element-Binding Protein; D010455:Peptides; C080549:CREBtide; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP