Figure 1. Phosphorylation of PKA substrates by cAMP analog and phosphatase inhibitor treatment. RAW 264.7 cells were either left untreated (-), treated with 10 nM CLA (+), or treated with various amounts of 8-Br-cAMP for 30 min and lysed in SDS sample buffer. The proteins were resolved on a 10% SDS gel, transferred to a nitrocellulose membrane, and immunoblotted with an anti-phospho-PKA substrates antibody (Phospho-(Ser/Thr) PKA substrates, Cell Signaling Technology). The mass of molecular weight markers (M) are shown at the left.

References• Coppolino MG, Krause M, Hagendorff, P, et al. (2001) J. Cell Sci. 114(23), 4307-4318.

• Krause M, Dent EW, Bear JE, et al. (2003) Annu. Rev. Cell Dev. Biol. 19, 541-564.

• Shabb JB (2001) Chem. Rev. 101(8), 2381-2411.

• Smolenski A, Bachmann C, Reinhard K, et al. (1998) J. Biol. Chem. 273(32), 20029-20035.

• Walders-Harbeck B, Khaitlina SY, Hinssen H, et al. (2002) FEBS Lett. 529, 275-280.

Identification and Validation of a PKA substrate in RAW 264.7 CellsEl Mazouni F, Bi Q, Lyons K, Draper L, Sethuraman D, Shu H, Brekken D

Alliance for Cellular Signaling Laboratories, University of Texas Southwestern Medical Center, Dallas, TX

Abstract

In its current form, the AfCS ligand screen does not include markers for activation of Protein Kinase A (PKA). While measurement of cAMP levels provides an indication of PKA activation, ligands that interact with PKA at the level of substrate phosphorylation are missed in the current screen. Inclusion of a PKA substrate in the ligand screen should provide information about the contributions of this pathway to macrophage signaling. Therefore, we carried out experiments to identify PKA substrates in RAW 264.7 cells. Proteins from RAW cells stimulated with a combination of 8‑Br‑cAMP and calyculin A, a serine/threonine phosphatase inhibitor, were immunoprecipitated with a phospho-PKA substrate antibody. Following either SDS-PAGE or further enrichment of the phosphopeptides using immobilized metal affinity chromatography (IMAC), the proteins were identified by liquid chromatography-tandem mass spectrometry. Vasodilator-stimulated phosphoprotein (VASP) was identified as one of several potential PKA substrates. To determine if VASP is a bona fide PKA substrate in RAW cells, we used phosphospecific anti-VASP antibodies to monitor VASP phosphorylation at two known PKA sites (Ser157 and Ser239). Stimulation with 8-Br-cAMP induced phosphorylation of both sites that was blocked by the PKA-selective inhibitor H89. Stimulation with 8-Br-cGMP (and presumably activation of PKG) did not cause phosphorylation of these sites. A time-course of VASP phosphorylation in response to stimulation by isoproterenol, sphingosine-1-phosphate, or the combination of both ligands was carried out. A robust phosphorylation of VASP was observed when cells were stimulated with the ligand combination (a ligand pair that causes substantial increases in cAMP). The time-course indicated that VASP phosphorylation was delayed by several minutes relative to the peak of ligand-stimulated cAMP production. The major conclusion is that VASP is a readily detectable PKA substrate in RAW cells. Even though reported to also be a PKG substrate in other systems, VASP does not appear to be phosphorylated at these sites by PKG in RAW cells.

Introduction

Cyclic AMP (cAMP) plays an important role in regulating cell function. Numerous cell surface receptors activate adenylyl cyclase to generate intracellular cAMP. Most of the actions of cAMP are mediated by cAMP-dependent protein kinase (PKA). PKA activation results in the phosphorylation of target proteins on serine or threonine residues in the context of a relatively well defined consensus sequence motif (RRXS/T). Substrates of PKA include a wide range of enzymes, ion channels, structural proteins, signaling proteins, and transcription factors (Shabb, 2001). The number of identified substrates has increased due to the availability of cell-permeable PKA-selective inhibitors, cell-permeable cAMP analogues, and phosphopeptide-specific antibodies. To identify markers of PKA activation for inclusion in the AfCS Ligand Screen, we set out to identify PKA substrates. PKA was activated with the cAMP analog 8-Br-cAMP and substrates isolated using the anti-phospho-PKA substrates antibody from Cell Signaling Technology.

Vasodilator-stimulated phosphoprotein (VASP) was originally identified as substrate of PKA in smooth muscle. VASP belongs to the Ena/VASP family of adaptor proteins that link the cytoskeleton to signal transduction. VASP functions in cytoskeleton organization, fibroblast migration, platelet activation, and axon guidance (Krause et al., 2003). In macrophages, VASP is reported to complex with Fyb/SLAP, SLP-76, Nck, and WASP to link cytoskeleton to FcR signaling during phagocytosis (Coppolino et al., 2001). VASP phosphorylation is reported to reduce its assoc with actin and has a negative effect on actin polymerization (Walders-Harbeck et al., 2002). VASP has three known phosphorylation sites (Ser157, Ser239, Thr278). Ser239 is reported to be the major PKG site while Ser157 is reported to be the major PKA site (Smolenski et al., 1998). To determine if VASP is a bona fide PKA substrate in RAW cells, we used phosphospecific anti-VASP antibodies to monitor VASP phosphorylation at Ser157 and Ser239.

ResultsConclusions

• VASP is a readily detectable PKA substrate in RAW cells.

• Phosphorylation of VASP by the combination of Iso+S1P shows a synergistic response that parallels the synergistic increase in cAMP production.

• VASP phosphorylation is delayed by several minutes relative to the peak of ligand-stimulated cAMP production.

• VASP does not appear to be phosphorylated by PKG in these cells.

• Unfortunately, the phospho-specific VASP antibodies from CST stain a large number of non-specific bands and therefore can not be added to the Ligand Screen antibody mixes.

Table 1. Proteins identified in 8-Br-cAMP+CL-A treated RAW 264.7 cells following immunoprecipitation with anti-PKA substrates antibody and enrichment of phosphopeptides by IMAC.

Figure 3. VASP phosphorylation in cells treated with cAMP or cGMP analogs. RAW264.7 cells were incubated in serum free media containing 100ug/ml BSA for 1 hr. They were either left untreated (-) or pre-treated for 1hr with 50 uM H89, a PKA inhibitor (H89). The cells were then either left untreated (C) or treated for 30 min with 2 mM 8-Br-cAMP (8-Br-cAMP) or 2 mM 8-Br-cGMP (8-Br-cGMP). The cells were lysed in SDS sample buffer. Aliquots of the lysate were resolved on 10% SDS gels. The gels were transferred to membranes and immunoblotted with anti-phospho-VASP (Ser157) Ab (#3111) (Panel A) or anti-phospho-VASP (Ser239) Ab from Cell Signaling Technology (#3114) (Panel B). The masses of molecular weight markers are shown to the left and the migration of VASP is shown by arrows on the right.

Figure 5. Time course of cAMP levels in comparison to VASP phosphorylation. The fold change in cAMP levels, shown on the left y-axis, upon treatment with 50 nM isoproterenol and 1 uM sphingosine-1-phosphate is plotted over time (pink line). On the right y-axis, the fold change in VASP Ser239 phosphorylation with the same treatment is plotted over time (blue line).

Methods

• RAW 264.7 cells were treated with 8-Br-cAMP, 8-Br-cGMP, calyculin A, PKA inhibitor H89, PKG inhibitor KT5823, isoproterenol and/or sphingosine-1-phosphate.

• PKA substrates were enriched by immunoprecipitation using a phospho-PKA substrates antibody (Cell Signaling Technology).

• A portion of the enriched proteins were separated on SDS gels and stained with Colloidal Blue. Individual bands were excised and proteins digested with trypsin.

• Alternatively, the enriched proteins were digested with trypsin and the phosphopeptides were further enriched using IMAC.

• Proteins were identified by LC-MS/MS using a nanoscale C18 column coupled in-line with an ion trap mass spectrometer. The MS and MS/MS data were used to search NCBI mouse and mammalian nonredundant protein databases to identify proteins.

• Vasodilator-stimulated phosphoprotein (VASP) phosphorylation was monitored by western blotting with phospho-specific antibodies to two distinct VASP Ser residues.

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Figure 2. Enrichment of phospho-PKA substrates from cells treated with 8-Br-cAMP and calyculin A. RAW264.7 cells were incubated in serum free media containing 100ug/ml BSA for 1 hr. They were then treated for 1 hr without (Ctrl) or with 2 mM 8-Br-cAMP and 50 nM calyculin A (8-Br+CLA). The cells were lysed in Lysis buffer containing 0.5% NP-40 and the lysate was cleared by centrifugation at 18,000 x g. The lysate was incubated overnight with an anti-phospho-PKA substrates antibody from CST and protein A Affi-Prep resin. The beads were washed with lysis buffer and the proteins were eluted with 100mM TEA pH 11.0. Aliquots of the eluate were resolved on a 12.5% SDS gel. The gel was stained with Colloidal Coomassie stain. The masses of molecular weight markers (M) are shown to the right and the bands excised and identified are numbered 1-4.

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Figure 4. Time course of VASP phosphorylation in cells treated with Iso and/or S1P. RAW264.7 cells were incubated in serum free media containing 100ug/ml BSA for 1 hr. They were incubated for 1, 2, 4, 6, 10, or 20 min with either media (Control), 50 nM isoproterenol (Iso), 1 uM sphingosine-1-phosphate (S1P) or with both Iso and S1P simultaneously (Iso+S1P). The cells were lysed in SDS sample buffer. Aliquots of the lysate were resolved on an SDS gel, transferred to a membrane, and immunoblotted with an anti-phospho-VASP (Ser239) Ab and an anti-RhoGDI Ab for a loading control (Panel A). The masses of molecular weight markers and the migration of VASP and RhoGDI are shown. After standardization of protein loading using the RhoGDI band, the fold change of the phospho-VASP band intensity was calculated in comparison to the control for the given time point. The fold change in VASP phosphorylation for the various treatments is plotted over time in Panel B.

Figure 7. VASP complex links the actin cytoskeleton to Fc receptor-mediated phagocytosis. SLP-76 interacts with Fyb/SLAP to recruit VASP and profilin. SLP-76 can also interact with Nck, which recruits WASP and Arp2/3 to the site of phagocytosis (Coppolino et al., 2001). Phosphorylation of VASP has been reported to decrease its ability to bind G-actin (Walders-Harbeck et al., 2002)

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Figure 6. Inhibition of VASP phosphorylation. RAW264.7 cells were incubated in serum free media containing 100ug/ml BSA for 30 min. They were either left untreated or pre-treated for 30 min with 50 uM H89, a PKA inhibitor (H89) or 10 uM KT5823, a PKG inhibitor (KT5823). They were then incubated for 1, 2, or 4 min with either media (Control), 50 nM isoproterenol (Iso), or with both Iso and 1 uM sphingosine-1-phosphate simultaneously (Iso+S1P). The cells were lysed in SDS sample buffer. Aliquots of the lysate were resolved on an SDS gel, transferred to membranes, and immunoblotted with either an anti-phospho-VASP Ser157 (Panel A) or phospho-VASP Ser239 (Panel B) Ab. The masses of molecular weight markers and the migration of VASP are shown.

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Protein name GI #Peptide sequence(found seq underlined)

Phos Site #

Scansite prediction for kinases phosphorylating identified site

Scansite PredictedPKA Site

Similar to Acyl-Coenzyme A binding domain containing 4

26359794 PQPLKQRS*PRRTR S259 Cdk5 (0.1828); Cdc2 (0.2320) Yes (T103)

DNA methyltransferase MmuI 20141336 KLESHT*VPVQSR T333 p38 MAPK (0.0875) Yes (S254, T324)

DNA methyltransferase MmuI 20141336 VPALAS*PAGSLPDHVR S15 p38 MAPK (0.0875) Yes (S254, T324)

Heterogeneous nuclear ribonucleoprotein U

17390825 AAGKSS*GPTSLFAVTVAPPGAR S183 GSK3 (0.7863) Yes (S26)

Lamin A 1794159 RSFRS*VGGSGGGSFGDNLVTR S99 None Yes (T10)

Lamin A/C9506843 LRLS*PSPT*SQR

S278PKA (0.1637); Cdk5 (0.2722), Cdc2 (0.3316); Erk1 (0.4142)

Yes (T45, S278)

Lamin C 91032 SGAQASSTPLS*PTRITRL S22p38 MAPK (0.0929); Cdk5 (0.1872); Cdc2 (0.2262); Erk1 (0.3288)

Yes (T10)

similarity to Mitochondrial Solute Carrier protein 38328284 RDFY*WLR

Y35 Itk (0.1396) No

Nucleolin 31543315 ALVPT*PGKKT121

p38 MAPK (0.1135); Cdk5 (0.2057); Cdc2 (0.2690)

Yes (T57)

Raly protein 13436047 GRLS*PVPVPRS119

PKA (0.1750); Cdk5 (0.2752); Cdc2 (0.3347); Erk1 (0.3417)

Yes (S119, T135)

Ribosomal protein L7 31981515 VATVPGT*LKKKVP T17PKC zeta (0.3607); PKC alpha/beta/ gamma (0.4554); Cdk5 (0.2793); Cdc2 (0.3303)

No

Vasodilator-stimulated phosphoprotein (VASP)

4322419 KLRKVS*KQEEASGGPLAPK S235 PKA (0.1230); Akt (0.4653) Yes (S153, S235, T274)

Iso+S1P cAMP and VASP(S239) phosphorylation

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