Research ArticlePSEUDOKINASES

The pseudokinase domains of guanylyl cyclase–A and –B allosterically increase the affinity of their catalytic domains for substrate

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Science Signaling  29 Jan 2019:
Vol. 12, Issue 566, eaau5378
DOI: 10.1126/scisignal.aau5378
  • Fig. 1 The R- and C-spines and residues that directly bind and transmit the allosteric ATP-binding signal in PKA are conserved in the PKD of GC-A.

    (A) Structure of the active site of the kinase domain of PKA and the LCK-based homology model of the corresponding site in the PKD of GC-A showing conservation of the residues (red) that interact with ATP (blue) in the active site. The catalytic base (Asp166) in PKA is not present in GC-A, where this residue is replaced with Asn628 (yellow). (B) Structures showing the R-spine (red), the C-spine (green), and the F helix (yellow) in the kinase domain of PKA and the homology model of the PKD of GC-A. (C) The serine and threonine residues mutated in nonphosphorylated or phosphomimetic constructs are indicated with their position numbers. Residues in red are phosphorylation sites that were chemically determined (8), and those in blue were identified by a functional screen (13).

  • Fig. 2 Structural alignment of the PKD of GC-A with crystal structures from other kinases.

    The homology model of the PKD of GC-A and the structures of PKA, BRAF, and LCK were aligned in PyMol. A linear representation of this structural alignment is shown with secondary structure features annotated above the alignment. α, alpha helix; β, beta sheet; Act Loop, activation loop. Residues comprising the R-spine (red) and the C-spine (cyan) and those that interact with ATP (gray) are highlighted. See also Table 1.

  • Fig. 3 Decreased NP-dependent GC activity in Lys and Asp mutants is not explained by changes in protein abundance or phosphorylation.

    (A) GC activity of membranes from HEK293T cells expressing the indicated wild-type (WT) and mutant forms of GC-A in the presence of GTP, ANP, ATP, and MgCl2 (ANP + ATP) or Triton X-100 and MnCl2 (Mn2+ + Triton). n = 8 independent experiments. (B) GC activity of membranes from HEK293T cells expressing the indicated WT and mutant forms of GC-B in the presence of GTP, CNP, ATP, and MgCl2 (CNP + ATP) or Triton X-100 and MnCl2 (Mn2+ + Triton). n = 3 independent experiments. (C) ProQ Diamond Phosphopreotein Gel Stain and SYPRO Ruby Protein Gel Stain of immunoprecipitated (IP) WT and mutant GC-A and immunoblot (IB) of the same samples used for GC activity assays in (A). The arrowhead indicates the fully processed form of GC-A. Data are representative of n = 7 independent experiments. (D) ProQ Diamond Phosphopreotein Gel Stain and SYPRO Ruby Protein Gel Stain of immunoprecipitated WT and mutant GC-B and immunoblot of the same samples used for GC activity assays in (B). The arrowhead indicates the fully processed form of GC-B. Data are representative of n = 3 independent experiments. (E) Quantification of experiments in (C). n = 7 independent experiments. (F) Quantification of experiments in (D). n = 3 independent experiments. Error bars represent the SEM. *P < 0.05, ***P < 0.005, and ****P < 0.001.

  • Fig. 4 The GC-B-K551A mutant has reduced CNP-dependent enzymatic activity that is not explained by changes in phosphorylation.

    (A) GC activity of membranes from HEK293T cells expressing the indicated WT and mutant forms of GC-B in the presence of GTP, CNP, ATP, and MgCl2 (CNP + ATP) or Triton X-100 and MnCl2 (Mn2+ + Triton). n = 5 independent experiments. (B) Immunoblot (IB) showing GC-B in samples from (A). The arrowhead indicates the fully processed form of GC-B with GC activity (38). Data are representative of n = 3 experiments. Error bars represent the SEM. ***P < 0.005 and ****P < 0.001.

  • Fig. 5 Conserved Lys and Asp residues in PKDs are required for activation of GC-A and GC-B by ATP but not by NP.

    (A) GC activity of membranes from HEK293T cells expressing GC-A-WT at the indicated time points after adding GTP in the presence of MgCl2 (basal), ANP plus MgCl2 (ANP), or ANP, ATP, and MgCl2 (ANP + ATP). Basal and ANP activities are shown in the inset. n = 3 independent experiments. (B) GC activity of membranes from HEK293T cells expressing GC-B-WT at the indicated time points after adding GTP in the presence of MgCl2 (basal), CNP and MgCl2 (CNP), or CNP, ATP, and MgCl2 (CNP + ATP). Basal and ANP activities are shown in the inset. n = 3 independent experiments. (C to F) GC activity measurements as in (A) and (B) for membranes from cells expressing GC-A-K535A (C), GC-B-K551A (D), GC-A-D646A (E), or GC-B-D662A (F). n = 3 independent experiments. (G) Fold stimulation of WT and mutant GC-A GC activity by ATP calculated from (A), (C), and (E) and plotted as a function of time. n = 3 independent experiments. (H) Fold stimulation of WT and mutant GC-B GC activity by ATP calculated from (B), (D), and (F) and plotted as a function of time. n = 3 independent experiments. (I) Fold stimulation of WT and mutant GC-A GC activity by ANP calculated from (A), (C), and (E) and plotted as a function of time. n = 3 independent experiments. (J) Fold stimulation of WT and mutant GC-B GC activity by CNP calculated from (B), (D), and (F) and plotted as a function of time. n = 3 independent experiments. Error bars represent the SEM. *P < 0.05, ***P < 0.005, and ****P < 0.001.

  • Fig. 6 A GC-A mutant lacking the PKD contains a cooperative, ATP-binding, allosteric site.

    (A and B) GC activity in membranes from Cos7 cells expressing a form of GC-A in which the PKD domain was deleted and in the presence of ANP, MgCl2, and increasing concentrations of GTP with or without the addition of ATP. n = 4 independent experiments. The dashed area in (A) is enlarged in (B). n = 4 independent experiments. (C) A table showing the measured Hill slope (h) and Khalf of the plots in (A) and (B) in the absence and presence of ATP and their respective P values. Error bars represent the SEM.

  • Fig. 7 Lys and Asp mutations increase the Michaelis constants for GC-A and GC-B.

    (A) GC activity of membranes from HEK293T cells expressing GC-A-WT in the presence of MgCl2, ANP, and increasing concentrations of GTP with or without ATP. n = 4 independent experiments. (B) GC activity of membranes from HEK293T cells expressing GC-B-WT in the presence of MgCl2, CNP, and increasing concentrations of GTP with or without ATP. n = 3 independent experiments. (C to F) GC activity as in (A) and (B) for membranes from cells expressing GC-A-K535A (C), GC-B-K551A (D), GC-A-D646A (E), or GC-B-D662A (F). n = 3 or 4 independent experiments. (G) A table showing the measured Michaelis constants of the indicated WT and mutant forms of GC-A and GC-B in the absence and presence of ATP and their associated P values. Error bars represent the SEM.

  • Fig. 8 The GC-A-A533W mutation partially mimics the ATP-bound state of GC-A.

    (A) A model demonstrating how ATP (orange) rigidifies the C-spine (green) in PKA and how the Trp in the GC-A-A533W mutant (yellow) docks into the same pocket. The R-spine is shown in red. (B) Substrate-velocity GC assays in membranes from HEK293T cells expressing GC-A-WT or GC-A-A533W in the presence of MgCl2, ANP, and increasing concentrations of GTP with or without ATP. n = 4 independent experiments. (C) A table showing the measured Michaelis constants and their associated P values. Error bars represent the SEM.

  • Fig. 9 The GC-B-M571F mutation increases GC activity at subsaturating concentrations of CNP.

    (A) A homology model showing the location of M571F in the C helix. Met571 is located in the C helix (gray) and contributes to the R-spine in position 3 (RS3). The R-spine residues RS2, RS3, and RS4 are shown in red. The M571F mutant is shown in yellow. (B) GC activity in membranes from HEK293T cells expressing GC-B-WT and GC-B-M571F in the presence of GTP, MgCl2, and ATP with the indicated concentrations of CNP. Basal, no CNP. n = 3 independent experiments. Inset: Immunoblot for GC-B in the samples used for GC assays. The arrowhead indicates the fully processed form of GC-B indicative of GC activity (38). n = 3 independent experiments. Error bars represent the SEM. **P < 0.01.

  • Fig. 10 The GC-B-I583W mutation increases stimulation by either ATP or ANP alone.

    (A) A homology model of GC-A shows the location of the GC-A-V567 W substitution that is analogous to the GC-B-I583W substitution. Yellow spheres represent the substituted Trp. This model shows how the Trp bridges the R-spine (red) and C-spine (green) to increase the activity of GC-B in the absence of either CNP or ATP. (B) GC activity of membranes from HEK293T cells expressing GC-B-WT or GC-B-I583W in the presence of GTP and MgCl2, under the indicated conditions. For Mn2+ + Triton conditions, MnCl2 was substituted for MgCl2. n = 3 independent experiments. Error bars represent the SEM. **P < 0.01 and ****P < 0.001.

  • Table 1 Amino acids that are important for ATP binding and allosteric transmission of the ATP-binding signal are conserved between PKA and GC-A and GC-B.

    PKA is a representative example of serine-threonine kinases. BRAF is a representative example of Tyr-like serine-threonine kinases, and LCK is a representative example of Tyr kinases and was the source of the homology model.

    PKABRAFLCKGC-AGC-B
    ATP-interacting amino acids
    K72K483K273K535K551
    E91E501E288E551E567
    D166D576D364N628S644
    K168K578R366K630K646
    N171N581N369N633N649
    D184D594D382D646D662
    R-spine
    L106 (RS4)F516L303F566F582
    L95 (RS3)L505M292M555M571
    F185 (RS2)F595F383Y647Y663
    Y164 (RS1)H574H362H626H642
    D220 (RS0)D638D422D689D706
    C-spine
    V57V471V259L511L527
    A70A481A271A533A549
    M128L537L324L588L604
    L172I582I370C634C650
    L173F583L371V635V651
    I174L584V372V636V652
    L227V601L429I696I713
    M231L605I433I700I717

Supplementary Materials

  • This PDF file includes:

    • Fig. S1. 8-Azido-2′/3′-biotinyl-ATP binding.
    • Fig. S2. ATP-agarose interaction assay.

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