Abstract
General strategies to obtain inactive kinases have utilized mutation of key conserved residues in the kinase core, and the equivalent Lys72 in cAMP-dependent kinase has often been used to generate a "dead" kinase. Here, we have analyzed the consequences of this mutation on kinase structure and function. Mutation of Lys72 to histidine (K72H) generated an inactive enzyme, which was unphosphorylated. Treatment with an exogenous kinase (PDK-1) resulted in a mutant that was phosphorylated only at Thr197 and remained inactive but nevertheless capable of binding ATP. Ser338 in K72H cannot be autophosphorylated, nor can it be phosphorylated in an intermolecular process by active wild type C-subunit. The Lys72 mutant, once phosphorylated on Thr197, can bind with high affinity to the RIalpha subunits. Thus a dead kinase can still act as a scaffold for binding substrates and inhibitors; it is only phosphoryl transfer that is defective. Using a potent inhibitor of C-subunit activity, H-89, Escherichia coli-expressed C-subunit was also obtained in its unphosphorylated state. This protein is able to mature into its active form in the presence of PDK-1 and is able to undergo secondary autophosphorylation on Ser338. Unlike the H-89-treated wild type protein, the mutant protein (K72H) cannot undergo the subsequent cis autophosphorylation following phosphorylation at Thr197. Using these two substrates and mammalian-expressed PDK-1, we can elucidate a possible two-step process for the activation of the C-subunit: initial phosphorylation on the activation loop at Thr197 by PDK-1, or a PDK-1-like enzyme, followed by second cis autophosphorylation step at Ser338.
Highlights
Most protein kinases are themselves phosphoproteins that contain an essential phosphate in the activation loop of the enzyme (1–5)
Nothing is known about the conformation of the C-subunit in its unphosphorylated state, and very little is known about the process by which the inactive dephosphorylated protein is converted into a fully phosphorylated protein prior to its association with regulatory subunits
Using phospho-specific antibodies, we show that both H-89 and the Lys72 mutants are excellent substrates for PDK-1 at Thr197; only wild type C-subunit can be phosphorylated at Ser338 and converted into an active enzyme
Summary
Materials—Reagents were obtained as follows: pRSETB expression vector (Invitrogen), [␥-32P]ATP (PerkinElmer Life Sciences), Escherichia coli strains BL21(DE3) (Novagen, Madison, WI), H-89 (LC Laboratories, Woburn, MA), Muta-Gene site directed mutagenesis kit and Affi-Gel (Bio-Rad), horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Biosciences), Gammabind G-Sepharose (Amersham Biosciences), pcDNA-3 eukaryotic expression vector (Invitrogen), SuperSignal West Pico chemiluminescent substrate detection kit (Pierce), oligonucleotides (Genosis-Sigma), the PepTag PKA activity assay kit (Promega, Madison, WI), and Effectene transfection kit (Qiagen, Valencia, CA). Lysed bacterial supernatant expressing the wild type or mutant proteins was incubated with the tagged Kemptide substrate and activator buffers at 30 °C, and the reaction was run on a 1% agarose gel at 100 V. Aliquots with and without the addition of wild type enzyme were added to SDS-gel loading buffer and subjected to SDSPAGE followed by immunoblotting using the ␣-C-subunit and ␣-Ser338-P antibodies to determine the phosphorylation state of Ser338. Phosphorylation State of Transfected C-subunit Constructs—Wild type, T197A, and S338A in the pcDNA-3 expression vector HA-tagged at the C terminus were transfected into COS cells using the Effectine transfection kit as per the manufacturer’s protocol. Transfected C-subunit was isolated by immunoprecipitation using a mouse monoclonal anti-HA antibody These samples were immunoblotted with the indicated rabbit generated antibodies to determine expression and phosphorylation state
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