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. 2006 Mar 14;45(10):3396-403.
doi: 10.1021/bi052513u.

Csk mediates G-protein-coupled lysophosphatidic acid receptor-induced inhibition of membrane-bound guanylyl cyclase activity

K S Madhusoodanan  1 , Dagang Guo, Deirdre K McGarrigle, Thomas Maack, Xin-Yun Huang
Affiliations

Csk mediates G-protein-coupled lysophosphatidic acid receptor-induced inhibition of membrane-bound guanylyl cyclase activity

K S Madhusoodanan et al. Biochemistry. .

Abstract

Natriuretic peptides (NPs) are involved in many physiological processes, including the regulation of vascular tone, sodium excretion, pressure-volume homeostasis, inflammatory responses, and cellular growth. The two main receptors of NP, membrane-bound guanylyl cyclases A and B (GC-A and GC-B), mediate the effects of NPs via the generation of cGMP. NP-stimulated generation of cGMP can be modulated by intracellular processes, whose exact nature remains to be elucidated. Thus, serum and lysophosphatidic acid (LPA), by unknown pathways, have been shown to inhibit the NP-induced generation of cGMP. Here we report that the nonreceptor-tyrosine-kinase Csk is an essential component of the intracellular modulation of atrial natriuretic peptide (ANP)-stimulated activation of GC-A. The genetic deletion of Csk (Csk(-)(/)(-)) in mouse embryonic fibroblasts blocked the inhibitory effect of both serum and LPA on the ANP-stimulated generation of cGMP. Moreover, using a chemical rescue approach, we also demonstrate that the catalytic activity of Csk is required for its modulatory function. Our data demonstrate that Csk is involved in the control of cGMP levels and that membrane-bound guanylyl cyclases can be critically modulated by other receptor-initiated intracellular signaling pathways.

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Figures

Figure 1
Figure 1
LPA and serum do not exert an inhibitory effect on ANP-stimulated cGMP synthesis in Csk−/− cells. A. Elevation in the intracellular cGMP levels in MEF cells in response to ANP and CNP treatment. MEF cells were cultured in 6 well dishes to 80% confluence. After serum starvation for 18–24 hours, cells were treated with starvation medium containing 0.5 mM IBMX for 30 minutes at 37°C, followed by treatment with 200 nM ANP or 200 nM CNP for 3 min at 37°C. Cells were washed once with PBS, lysed with 0.5% Triton X-100 containing 0.5 mM IBMX and cGMP content was determined. B. LPA (10 μM) and serum (10% FBS) attenuate ANP induced cGMP synthesis in MEF cells. C. ANP response is enhanced, and LPA and serum do not exert an inhibitory effect on ANP-stimulated cGMP synthesis in Csk−/− cells. Data represent the means ± S.D. of three experiments.
Figure 2
Figure 2
Transient re-expression of Csk in Csk−/− cells results in attenuation of ANP stimulated cGMP production and reconstitution of LPA inhibition. Csk−/− cells grown to 70%–80% confluence in 6 well dishes was transiently transfected with a vector plasmid (Column 1) or plasmids carrying human Csk (Columns 2 and 3) as described in experimental protocols. 16–18 hours after transfection, cells were serum starved overnight and treated for 30 min at 37°C and 5% CO2 with medium containing LPA and 0.5 mM IBMX. This was followed by treatment with 200 nM ANP at 37°C for 3 min. Medium was aspirated, cells were washed once with PBS, lysed with 0.5% Triton X-100 containing 0.5 mM IBMX and cGMP present in the samples was determined. * indicates that the cGMP level from Csk−/−+Csk+ANP is significant different from that from Csk−/−+Csk+ANP+LPA (t-test: p<0.05). The values shown represent the means ± S.D. of three experiments.
Figure 3
Figure 3
The catalytic activity of Csk is essential for its regulation of receptor guanylyl cyclase activity. A. Csk−/− cells stably expressing the CskR318A mutant were grown to 80% confluence in 6 well dishes. Following serum starvation overnight, cells were treated with medium containing LPA and 0.5 mM IBMX for 30 min at 37°C in the presence (▼) or absence (■) of 50 mM imidazole. This was followed by treatment with indicated concentrations of ANP for 3 min at 37°C. Cells were then washed once with PBS, lysed with 0.5% Triton X-100 containing 0.5 mM IBMX and cGMP content present in the samples was determined. B. Treatment of Csk−/− cells with imidazole (Column 3) or transient expression of CskR318A in Csk−/− cells (without imidazole addition) (Column 4) did not lead to significant changes in ANP-stimulated cGMP production. Data are representative of three similar experiments.
Figure 4
Figure 4
Attenuation of ANP-stimulated cGMP synthesis by Csk does not require PKC activity. A. MEF cells were serum-starved overnight. After pretreatment with Calphostin C or vehicle, cells were treated with ANP, LPA then ANP, or PMA then ANP for 3 min. Cells were then washed once with PBS, lysed with 0.5% Triton X-100 containing 0.5 mM IBMX and cGMP content present in the samples was determined. B. Csk−/− cells grown to 70–80% confluence in 6 well dishes were transiently transfected with CskR318A or empty vector (pcDNA3.1). 16–18 hours after transfection, cells were serum-starved overnight. After pretreatment with Calphostin C or vehicle, cells were treated with growth medium containing 50 mM imidazole and 0.5 mM IBMX for 30 min at 37°C. This was followed by treatment with 200 nM ANP for 3 min at 37°C and 5% CO2. Cells were then washed once with PBS, lysed with 0.5% Triton X-100 containing 0.5 mM IBMX and cGMP content present in the samples was determined. Data represent the means ± S.D. of three experiments.
Figure 5
Figure 5
Csk does not directly phosphorylate GC-A. A. Coomassie blue staining of purified recombinant Csk, Src and GST-GC-A intra (the intracellular domain of GC-A). B. In vitro kinase assay of Csk using GST-CDB3 and GST-GC-A as exogenous substrates. The in vitro kinase assay was performed in the presence of ATP and the results were analyzed with SDS-PAGE and western blotted with anti-phospho-Tyr antibody. C. In vitro kinase assay of Src using GST-CDB3 and GST-GC-A-intra as substrates. The kinase assay was performed in the presence of γ-32P-ATP and the results were analyzed by SDS-PAGE and autoradiography.
Figure 6
Figure 6
LPA-induced decrease of Ser/Thr phosphorylation of GC-A depends on Csk. A. Csk−/−/Csk cells were serum-starved. Cells were then treated with LPA or PMA. Cell lysates were immunoprecipitated with anti-GC-A antibody. After SDS-PAGE, the filters were probed with anti-phospho-Ser/Thr antibody (top panel) or anti-GC-A antibody (bottom panel). B. The intensity of each band in A was quantified by an image analyzer. The percentage of phosphorylated GC-A is corrected by the amount of the total GC-A. C. Csk−/− cells were serum-starved. Cells were then treated with LPA or PMA. Cell lysates were immunoprecipitated with anti-GC-A antibody. After SDS-PAGE, the filters were probed with anti-phospho-Ser/Thr antibody (top panel) or anti-GC-A antibody (bottom panel). D. The intensity of each band in C was quantified by an image analyzer. The percentage of phosphorylated GC-A is corrected by the amount of the total GC-A. * indicates an significant difference from the untreated cells (t-test: p<0.05). Data represent the means ± S.D. of three experiments.
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