Phosphorylation of the catalytic subunit of protein kinase A. Autophosphorylation versus phosphorylation by phosphoinositide-dependent kinase-1.
The identification of phosphoinositide-dependent kinase-1 (PDK-1) as an activating kinase for members of the AGC family of kinases has led to its implication as the activating kinase for cAMP-dependent protein kinase. It has been established in vitro that PDK-1 can phosphorylate the catalytic (C) subunit (), but the Escherichia coli-expressed C-subunit undergoes autophosphorylation. To assess which of these mechanisms occurs in mammalian cells, a set of mutations was engineered flanking the site of PDK-1 phosphorylation, Thr-197, on the activation segment of the C-subunit. Two distinct requirements appeared for autophosphorylation and phosphorylation by PDK-1. Autophosphorylation was disrupted by mutations that compromised activity (Thr-201 and Gly-200) or altered substrate recognition (Arg-194). Conversely, only residues peripheral to Thr-197 altered PDK-1 phosphorylation, including a potential hydrophobic PDK-1 binding site at the C terminus. To address the in vivo requirements for phosphorylation, select mutant proteins were transfected into COS-7 cells, and their phosphorylation state was assessed with phospho-specific antibodies. The phosphorylation pattern of these mutant proteins indicates that autophosphorylation is not the maturation mechanism in the eukaryotic cell; instead, a heterologous kinase with properties resembling the in vitro characteristics of PDK-1 is responsible for in vivo phosphorylation of PKA.
Moore
Michael J
MJ
Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0654, USA.
Kanter
Joan R
JR
Jones
K C
KC
Taylor
Susan S
SS
D064413:3-Phosphoinositide-Dependent Protein Kinases; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D019556:COS Cells; D020134:Catalytic Domain; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000737:chemistry; D014162:Transfection
D020033:Protein Isoforms; D013912:Threonine; D001120:Arginine; D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D005998:Glycine