A Screen for PKN3 Substrates Reveals an Activating Phosphorylation of ARHGAP18
Abstract
:1. Introduction
2. Results
2.1. Phosphoproteomic Screen for Novel PKN3 Substrates
2.2. ARHGAP18 is Phosphorylated by PKN3
2.3. PKN3 Interacts with ARHGAP18 via its N-Terminal Region
2.4. Phosphorylation of ARHGAP18 Isoform1 but not Isoform2, Promotes Interaction with PKN3
2.5. Phosphorylation of ARHGAP18 Leads to Activation of Its GAP Domain
3. Discussion
4. Materials and Methods
4.1. Cell Lines and Cell Cultivation
4.2. Plasmid Construction
4.3. Protein Expression and Purification
4.4. Screen for PKN3 Substrates, Sample Preparation and Data Analysis
4.5. Immunoprecipitation and Immunoblotting
4.6. Antibodies
4.7. Kinase Assays
4.8. RhoA-GTP Pull-Down Assay
4.9. RhoA Pull-Down Assay
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
PKN3 | Protein Kinase N3 |
ARHGAP18 | Rho GTPase Activating Protein 18 |
N6-Bn ATPγS | N6-Benzyl ATPγS |
PNBM | p-nitrobenzyl mesylate |
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Gene | Site | Surrounding Sequence | PLP | Score |
---|---|---|---|---|
ARHGAP18 | T158 | KRVETVSQTLRKKNKQY | 1.00 | 111.46 |
S156 | VQKRVETVSQTLRKKNK | 1.00 | 111.46 | |
BRD4 | S601 | SKPPPTYESEEEDKCKP | 1.00 | 101.9 |
CCDC144A | S805 | EMARKKMNSEISHRHQK | 0.99 | 82.029 |
CEACAM16 | T402 | NLTDTGRYTLKTVTVQG | 1.00 | 100.25 |
T398 | VQKLNLTDTGRYTLKTV | 1.00 | 100.25 | |
DCAF12L1 | S8 | _MAQQQTGSRKRKAPAV | 1.00 | 83.801 |
DSCAM | S1408 | LPGDNGGSSIRGYILQY | 0.98 | 87.498 |
IGSF22 | S318 | LSVGDKRMSAELTVLDE | 0.99 | 84.817 |
KIF27 | S754 | TGNDAKSVSKQYSLKVT | 1.00 | 120.77 |
T746 | DLIKELIKTGNDAKSVS | 0.99 | 120.77 | |
NFE2L2 | T137 | KLHHNYKITIYSM____ | 1.00 | 99.139 |
ODF1 | S10 | AALSCLLDSVRRDIKKV | 1.00 | 103.7 |
OR4K15 | T158 | ICKPLHYMTVMSRRVCV | 0.99 | 79.906 |
POLE1 | S891 | VKKPKVTISYPGAMLNI | 1.00 | 82.483 |
T889 | TNVKKPKVTISYPGAML | 1.00 | 82.483 | |
PRDM16 | S1218 | DVLNSTLDSEALKHTLC | 0.99 | 96.371 |
RETSAT | S322 | VLTKATVQSVLLDSAGK | 0.98 | 99.53 |
SACS | S1222 | GIFTKPSLSAVLKHFKI | 1.00 | 86.512 |
SPATA31D1 | T1339 | VLGSKSSPTLKTQPPPE | 0.87 | 86.189 |
USP42 | T786 | PRDPGTPATKEGAWEAM | 1.00 | 93.096 |
UTP20 | S926 | HLQVFSKFSNPRALYLE | 1.00 | 101.65 |
VPS33B | S476 | AGKITDAFSSLAKRSNF | 0.78 | 83.206 |
S477 | GKITDAFSSLAKRSNFR | 0.78 | 83.206 | |
VWA3B | T99 | EDGRVYNLTAKSELIYQ | 1.00 | 84.244 |
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Dibus, M.; Brábek, J.; Rösel, D. A Screen for PKN3 Substrates Reveals an Activating Phosphorylation of ARHGAP18. Int. J. Mol. Sci. 2020, 21, 7769. https://doi.org/10.3390/ijms21207769
Dibus M, Brábek J, Rösel D. A Screen for PKN3 Substrates Reveals an Activating Phosphorylation of ARHGAP18. International Journal of Molecular Sciences. 2020; 21(20):7769. https://doi.org/10.3390/ijms21207769
Chicago/Turabian StyleDibus, Michal, Jan Brábek, and Daniel Rösel. 2020. "A Screen for PKN3 Substrates Reveals an Activating Phosphorylation of ARHGAP18" International Journal of Molecular Sciences 21, no. 20: 7769. https://doi.org/10.3390/ijms21207769