Enhanced Dephosphorylation of cAMP-dependent Protein Kinase by Oxidation and Thiol Modification

Kenneth M Humphries,Michael S Deal,Susan S Taylor

doi:10.1074/jbc.m410242200

Kenneth M Humphries, Michael S Deal + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m410242200

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Abstract

The catalytic subunit of cAMP-dependent protein kinase (PKA) is phosphorylated at threonine 197 and serine 338. Phosphorylation of threonine 197, located in the activation loop, is required for coordinating the active site conformation and optimal enzymatic activity. However, this phosphorylation has not been widely appreciated as a regulatory site because of the apparent constitutive nature of the phosphorylation and the general resistance of the kinase to phosphatase treatment. We demonstrate here that the observed resistance of the catalytic subunit to dephosphorylation is due, in part, to the presence of the highly nucleophilic cysteine 199 located proximal to the phosphate on threonine 197. Experiments performed in vitro demonstrated that mutation (cysteine 199 to alanine), oxidation, such as by glutathionylation or internal disulfide bond formation, or alkylation of the C-subunit enhanced its ability to be dephosphorylated. Furthermore, rephosphorylation of reduced C-subunit by PDK1 created a cycle whereby the inactive kinase could be reactivated. To demonstrate that thiol modification of PKA can lead to enhanced dephosphorylation in vivo, PC12 cells were treated with N-ethylmaleimide (NEM). Such treatment resulted in complete PKA inactivation and dephosphorylation of threonine 197. This effect of NEM was contingent upon prior treatment of the cells with PKA activators, demonstrating the resistance of the holoenzyme to thiol alkylation-mediated dephosphorylation. Our results also demonstrated that NEM treatment of PC12 cells enhanced the dephosphorylation of the protein kinase Calpha activation loop, suggesting a common mechanism of regulation among members of the AGC family of kinases.

Highlights

CAMP-dependent protein kinase (PKA)1 is a ubiquitously expressed signaling molecule
We demonstrate here that the observed resistance of the catalytic subunit to dephosphorylation is due, in part, to the presence of the highly nucleophilic cysteine 199 located proximal to the phosphate on threonine 197
Our results demonstrated that NEM treatment of PC12 cells enhanced the dephosphorylation of the protein kinase C␣ activation loop, suggesting a common mechanism of regulation among members of the AGC family of kinases

Summary

EXPERIMENTAL PROCEDURES

Materials—All chemicals and reagents were obtained from Sigma Aldrich unless otherwise noted. The C-subunit (0.3 mg/ml) was incubated for 15 min with 100 ␮M diamide and 125 ␮M GSH at 23 °C in Buffer A followed by measurement of kinase activity. Alkylation of the kinase was performed by incubation of the kinase (0.3 mg/ml) with 250 ␮M Nethylmaleimide (NEM) for 20 min at 23 °C in Buffer A followed by the addition of 5.0 mM DTT. CAMP-dependent protein kinase activity was determined using the PepTag nonradioactive PKA assay system (Promega) [28]. For this assay, 7.0 ␮g of total cell lysate was added to the PepTag mixture containing 5.0 ␮M cAMP for 10 min, and the reaction was stopped by the addition of the PKA-specific inhibitor PKI (50 ␮M final). Following three washes for 5 min with TTBS, blots were incubated with SuperSignal West Pico chemiluminescent substrate (Pierce) for 5 min and exposed to HyperFilm (Amersham Biosciences) for 0 –5.0 min

RESULTS

Dephosphorylation of PKA

DISCUSSION