Differential sensitivity to inhibitors discriminates between two types of kinases responsible for in vivo phosphorylation of different sites in the carboxy-terminal tail of chicken neurofilament-M.
In order to characterize the phosphorylation of neurofilaments (NF) in intact neurons, we examined the ability of several protein kinase inhibitors to interfere with the incorporation 32P into individual NF polypeptides of sensory neurons in culture. We also examined their effect on the post-translational mobility shift on SDS-PAGE that accompanies phosphorylation of newly synthesized NF-M. Several agents known to inhibit cyclic nucleotide-, Ca2+/calmodulin-, and Ca2+/phospholipid-dependent protein kinases (H7, HA1004, trifluoperizine, sphingosine) had no effect on the phosphorylation of any NF polypeptide, in either assay. In contrast, two broadly active protein kinase inhibitors, staurosporine and K252a, inhibited the incorporation of 32P into NF-M by 60-70% and also blocked the post-translational mobility shift. They had no effect on NF-L. The action of staurosporine and K252a was identical to that of 25 mM LiCl. Proteolytic cleavage and phosphopeptide mapping of 32P-labeled NF-M from control and treated cultures revealed that the phosphorylation of only one subset of phosphopeptides was affected by staurosporine, K252a, and LiCl. These were contained within a single chymotryptic fragment of the NF-M tail segment, probably containing most of the 17 repeats of a KXXS/TP motif. The phosphorylation of another subset of phosphopeptides was insensitive to these inhibitors. They were contained within a different chymotryptic fragment of the tail segment which contains a KSD and four KSP potential phosphorylation sites. This differential sensitivity to protein kinase inhibitors distinguishes two different types of effector-independent kinases that phosphorylate, in vivo, different sites within the NF-M tail.
Bennett
G S
GS
Department of Anatomy, College of Medicine, University of Florida, Gainesville 32610.
Basu
U
U
Hollander
B A
BA
Quintana
R
R
Rodriguez
R
R
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D002642:Chick Embryo; D005727:Ganglia, Spinal / Q000166:cytology / Q000196:embryology; D026121:Indole Alkaloids; D007546:Isoquinolines / Q000494:pharmacology; D018021:Lithium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010879:Piperazines / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013110:Sphingosine / Q000494:pharmacology; D019311:Staurosporine; D014268:Trifluoperazine / Q000494:pharmacology
D000470:Alkaloids; D002227:Carbazoles; D026121:Indole Alkaloids; D007546:Isoquinolines; D016900:Neurofilament Proteins; D010879:Piperazines; C044882:neurofilament protein L; C061195:neurofilament protein M; D014268:Trifluoperazine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C049985:staurosporine aglycone; D017346:Protein Serine-Threonine Kinases; D018021:Lithium Chloride; D019311:Staurosporine; D013110:Sphingosine