Neuronal Cdc2-like protein kinase (Cdk5/p25) is associated with protein phosphatase 1 and phosphorylates inhibitor-2.
Protein phosphatase 1 (PP1) is complexed with inhibitor 2 (I-2) in the cytosol. In rabbit muscle extract PP1.I-2 is activated upon preincubation with ATP/Mg. This activation is caused by phosphorylation of I-2 on Thr(72) by glycogen synthase kinase 3 (GSK3). We have found that PP1.I-2 in bovine brain extract is also activated upon preincubation with ATP/Mg. However, blocking GSK3 action by LiCl inhibited only approximately 29% of PP1 activity and indicated that GSK3 is not the sole PP1.I-2 activator in the brain. When bovine brain extract was analyzed by gel filtration PP1.I-2 and neuronal Cdc2-like protein kinase (NCLK), a heterodimer of Cdk5 and the regulatory p25 subunit, co-eluted as a approximately 450-kDa size species. The NCLK from the eluted column fractions bound to PP1-specific microcystin-Sepharose and glutathione S-transferase (GST)-I-2-coated glutathione-agarose beads. Similarly, PP1 from the eluted column fractions was pulled down with GST-Cdk5-coated glutathione-agarose beads. In vitro, NCLK phosphorylated I-2 on Thr(72) and activated PP1.I-2 in an ATP/Mg-dependent manner. NCLK bound to PP1 through its Cdk5 subunit and the PP1 binding region was localized to Cdk5 residues 28-41. Our data demonstrate that in brain extract PP1.I-2 and NCLK are associated within a complex of approximately 450 kDa and suggest that NCLK is one of the PP1.I-2-activating kinases in the mammalian brain.
Agarwal-Mawal
A
A
Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Quebec, H3T 1E2, Canada.
Paudel
H K
HK
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002417:Cattle; D002457:Cell Extracts / Q000032:analysis; D002850:Chromatography, Gel; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000378:metabolism* / Q000502:physiology*; D004789:Enzyme Activation; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D054645:Protein Phosphatase 1; D011506:Proteins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D000097847:Protein Phosphatase Inhibitory Proteins
D000255:Adenosine Triphosphate; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002457:Cell Extracts; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D046911:Macromolecular Substances; D008274:Magnesium; D010749:Phosphoprotein Phosphatases; D010769:Phosphothreonine; D054645:Protein Phosphatase 1; D011506:Proteins; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins