The sites of phosphorylation of rabbit brain phosphofructo-1-kinase by cyclic AMP-dependent protein kinase.
The three isozymic subunits of phosphofructo-1-kinase present in rabbit brain and designated A, B and C were phosphorylated in vitro by cyclic AMP-dependent protein kinase with 32P-labeled ATP. Limited digestion of the labeled enzymes with trypsin or with Staphylococcus aureus V8 proteinase led to the solubilization of radiolabeled peptides derived from the three isozymic subunits. Limited digestion by V8 proteinase was accompanied by a slight reduction in the apparent sizes of the subunits, indicating that the phosphorylated sites are located near either the amino or carboxyl termini of the protein. V8 proteinase digestion led to no change in the maximal activity of the enzyme but did abolish sensitivity to ATP inhibition. The phosphopeptides of the tryptic and the V8 digests were purified by chromatography and their amino acid sequences were determined and compared to the previously established sequence from rabbit muscle isozyme A. PFK-A E H I S R K R S G E A T V PFK-B H V T R R S L S M A K G F PFK-C V S A S P R G S Y R K F L In each instance, the phosphorylated serine, underlined in the above sequences, was found to be one or two residues toward the C-terminus of one or more basic residues. No other similarities in structure were noted.
Valaitis
A P
AP
Department of Biological Chemistry and Structure, University of Health Sciences, Chicago Medical School, North Chicago, IL 60064.
Foe
L G
LG
Kwiatkowska
D
D
Latshaw
S P
SP
Kemp
R G
RG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP / Q000494:pharmacology*; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D007527:Isoenzymes; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D010732:Phosphofructokinase-1; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin