Mutations that alter the charge of type I regulatory subunit and modify activation properties of cyclic AMP-dependent protein kinase from S49 mouse lymphoma cells

Steinberg, Robert A.; Gorman, K B; Øgreid, D; Døskeland, Stein Ove; Weber, Irene T.

doi:10.1016/s0021-9258(19)67830-0

Cited by 31 publications

(5 citation statements)

References 35 publications

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“…A similar strategy was taken in characterizing binding pockets of PKA. 22,23 For the PKG Iβ mutants, our measurements show that disabling site A (G182E), thus having only intact site B, has no effect on its selectivity for PET, while slightly increasing its values for 8-Br-cGMP (from 328 nM to 765 nM). These values suggest that site B provides the selectivity for PET-cGMP.…”

Section: ■ Resultsmentioning

confidence: 86%

Structural Basis of Analog Specificity in PKG I and II

et al. 2017

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Cyclic GMP analogs, 8-Br, 8-pCPT, and PET-cGMP, have been widely used for characterizing cellular functions of cGMP-dependent protein kinase (PKG) I and II isotypes. However, interpreting results obtained using these analogs has been difficult due to their low isotype specificity. Additionally, each isotype has two binding sites with different cGMP affinities and analog selectivities, making understanding the molecular basis for isotype specificity of these compounds even more challenging. To determine isotype specificity of cGMP analogs and their structural basis, we generated the full-length regulatory domains of PKG I and II isotypes with each binding site disabled, determined their affinities for these analogs, and obtained cocrystal structures of both isotypes bound with cGMP analogs. Our affinity and activation measurements show that PET-cGMP is most selective for PKG I, whereas 8-pCPT-cGMP is most selective for PKG II. Our structures of cyclic nucleotide binding (CNB) domains reveal that the B site of PKG I is more open and forms a unique π/π interaction through Arg285 at β4 with the PET moiety, whereas the A site of PKG II has a larger β5/β6 pocket that can better accommodate the bulky 8-pCPT moiety. Our structural and functional results explain the selectivity of these analogs for each PKG isotype and provide a starting point for the rational design of isotype selective activators.

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Section: ■ Resultsmentioning

confidence: 86%

Structural Basis of Analog Specificity in PKG I and II

et al. 2017

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“…These residues form hydrogen-bond and ionic interactions with cAMP in CAP. Mutations of these residues reduce or eliminate cAMP binding to the altered site in cAPK (Steinberg et al, 1991;Shabb et al, 1990). Ala 84 is also conserved and forms part of the cyclic nucleotide binding pocket.…”

Section: Resultsmentioning

confidence: 99%

“…Helix B has conserved hydrophobic residues Leu 579, Ala 582, and Leu 583, and polar residues Asp 577, Asp 578, and Glu 581 at equivalent positions as in CAP and cGPK. The C helix has less sequence similarity and may be shorter than in CAP or in a different position as discussed for the cAMP-dependent protein kinase Steinberg et al, 1991). Internal Hydrophobic Packing.…”

Section: Resultsmentioning

confidence: 99%

“…The Thr 560 OH is predicted to interact with the 2-NH2 group, as in the cGMP-dependent protein kinase. Interactions with the 8-and 6-positions are more difficult to predict since these are close to the region corresponding to the C-helix in CAP, which may differ in the protein kinases as suggested by analysis of mutations (Steinberg et al, 1991). The C-helix region has little sequence similarity among the different proteins: CAP, kinases, and the ion channels.…”

Section: Resultsmentioning

confidence: 99%

“…Subsequently, the positions of most of the important cAMP-binding residues have been confirmed by sequencing of mutations with altered cAMP-binding properties (Bubis et al, 1988;Ogreid et al, 1988;Woodford et al, 1989;Correll et al, 1989). The models of the cyclic nucleotide binding domains of the protein kinases have proved useful in analysis and design of mutations (Steinberg et al, 1991;Shabb et al, 1990). In the present study, we have used the information from sequence similarities among these cyclic nucleotide receptors to construct a molecular model of the cyclic nucleotide-binding domain of the ion channel by analogy to the crystal structure of CAP with cAMP.…”

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confidence: 99%

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Molecular model of the cyclic GMP-binding domain of the cyclic GMP-gated ion channel

Kumar¹,

Weber²

1992

Biochemistry

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The structure of the cyclic GMP-binding domain of the cyclic GMP-gated ion channel from bovine retinal rod photoreceptors has been modeled by analogy to the crystal structure of the homologous cyclic AMP-binding domain of catabolite gene activator protein (CAP). The modeled cyclic GMP-binding domain has a three-residue deletion and a five-residue insertion between beta strands compared to CAP. The major interactions of the ion channel with cyclic GMP are similar to those observed for cyclic AMP bound to CAP and predicted for cGMP bound to the cGMP-dependent protein kinase: Gly 543 and Glu 544 make hydrogen-bond interactions with the ribose 2'-OH, Arg 559 forms an ion pair with the charged phosphate oxygen, and Thr 560 forms hydrogen-bond interactions with an exocyclic phosphate oxygen and with the 2-amino group of cGMP. Three additional potential interactions were predicted from the model structure. Ile 545 O and Ser 546 OH form hydrogen-bond interactions with an exocyclic phosphate oxygen, and Phe 533 may interact with the aromatic ring of cGMP. This model is in agreement with both the analogue binding experiments and the mutational analysis of Thr 560.

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Analysis of the dominance of mutations in cAMP‐binding sites of murine type I cAMP‐dependent protein kinase in activation of kinase from heterozygous mutant lymphoma cells

Shuntoh

Steinberg

1991

Journal Cellular Physiology

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Structural lesions in cAMP-binding sites of regulatory (R) subunit of cAMP-dependent protein kinase caused identical increases in apparent constants for cyclic nucleotide-dependent kinase activation in preparations from cells that were hemizygous or heterozygous for mutant R1 subunit expression. No wild-type kinase activation was observed in extracts from heterozygous mutant cells. This "dominance" was investigated by characterizing expression of wild-type and mutant R1 subunits and properties of protein kinase from S49 mouse lymphoma cell mutants heterozygous for expression of wild-type R1 subunits and R1 subunits with a lesion (Glu200) that inactivates cAMP-binding site A. By both studies of cAMP dissociation and two-dimensional gel analysis, wild-type R subunits comprised about 35% of total R1 subunits in heterozygous mutants. Synthesis of wild-type and mutant R1 subunits was equivalent, but wild-type subunits were degraded preferentially. Hydroxylapatite chromatography revealed a novel R1 subunit-containing species from heterozygous mutant preparations whose elution behavior suggested a trimeric kinase consisting of an R1 subunit dimer and one catalytic (C) subunit. Wild-type R1 subunit was found only in dimer and "trimer" peaks; the tetrameric kinase peak contained only mutant R1 subunit. It is concluded that C subunit binds preferentially to mutant R1 subunit in heterozygous cells forming either tetrameric kinase with mutant R1 subunit homodimers or trimeric kinase with R1 subunit heterodimers. This preferential binding results both in suppression of wild-type kinase activation and differential stabilization of mutant R1 subunits.

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Mutations that alter the charge of type I regulatory subunit and modify activation properties of cyclic AMP-dependent protein kinase from S49 mouse lymphoma cells

Cited by 31 publications

References 35 publications

Structural Basis of Analog Specificity in PKG I and II

Structural Basis of Analog Specificity in PKG I and II

Molecular model of the cyclic GMP-binding domain of the cyclic GMP-gated ion channel

Analysis of the dominance of mutations in cAMP‐binding sites of murine type I cAMP‐dependent protein kinase in activation of kinase from heterozygous mutant lymphoma cells

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