Properties of several protein kinases that copurify with rat spinal cord neurofilaments.
Several protein kinases that copurify with neurofilaments (NF) were identified and each kinase was assessed for its ability to phosphorylate NF proteins. NFs were isolated using an axonal flotation procedure and the kinases were extracted from NFs with 0.8 M KCl. NF kinases were incubated with peptide substrates for selected protein kinases, [32P]ATP and protein kinase cofactors and inhibitors to characterize the kinases. Using peptide substrates, three types of kinase were identified, and a fourth was identified using NF protein as substrate. The first three kinases were the catalytic subunit of cAMP-dependent protein kinase, calcium-calmodulin dependent protein kinase II and a cofactor-independent kinase that phosphorylated prepro VIP sequence 156-170 and was inhibited by heparin. Using NF proteins as substrate, a fourth kinase was identified which was cofactor-independent and was not inhibited by heparin. Neither cofactor-independent kinase was casein kinase II. NF proteins were phosphorylated in vitro on serine and threonine, primarily by the two cofactor-independent kinases. Using [alpha-32P]8-N3ATP for affinity labeling, one kinase of 43,800 Da was identified. Thus, in addition to cAMP-dependent protein kinase and calcium-calmodulin dependent protein kinase II, two kinases have been found which are primarily responsible for NF phosphorylation in vitro and are cofactor-independent.
Department of Pharmacology, ICI Pharmaceuticals Group, Wilmington, DE 19897.
C B
CB
Caputo
L A
LA
Sygowski
W F
WF
Brunner
C W
CW
Scott
A I
AI
Salama
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002746:Chlorpromazine / Q000494:pharmacology; D003599:Cytoskeleton / Q000201:enzymology*; D007381:Intermediate Filament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007382:Intermediate Filaments / Q000201:enzymology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013116:Spinal Cord; D014268:Trifluoperazine / Q000494:pharmacology; D014404:Tubulin / Q000378:metabolism
D000345:Affinity Labels; D002364:Caseins; D007381:Intermediate Filament Proteins; D010455:Peptides; D047428:Protein Kinase Inhibitors; D014404:Tubulin; D014268:Trifluoperazine; D011494:Protein Kinases; D002746:Chlorpromazine