The KLDpT activation loop motif is critical for MARK kinase activity

Autoři: Tim Sonntag aff001;  James J. Moresco aff002;  John R. Yates, III aff002;  Marc Montminy aff001
Působiště autorů: Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California, United States of America aff001;  Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, United States of America aff002
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article


MAP/microtubule-affinity regulating kinases (MARK1-4) are members of the AMPK family of Ser/Thr-specific kinases, which phosphorylate substrates at consensus LXRXXSXXXL motifs. Within microtubule-associated proteins, MARKs also mediate phosphorylation of variant KXGS or ζXKXGSXXNΨ motifs, interfering with the ability of tau and MAP2/4 to bind to microtubules. Here we show that, although MARKs and the closely related salt-inducible kinases (SIKs) phosphorylate substrates with consensus AMPK motifs comparably, MARKs are more potent in recognizing variant ζXKXGSXXNΨ motifs on cellular tau. In studies to identify regions of MARKs that confer catalytic activity towards variant sites, we found that the C-terminal kinase associated-1 (KA1) domain in MARK1-3 mediates binding to microtubule-associated proteins CLASP1/2; but this interaction is dispensable for ζXKXGSXXNΨ phosphorylation. Mutational analysis of MARK2 revealed that the N-terminal kinase domain of MARK2 is sufficient for phosphorylation of both consensus and variant ζXKXGSXXNΨ sites. Within this domain, the KLDpT activation loop motif promotes MARK2 activity both intracellularly and in vitro, but has no effect on SIK2 activity. As KLDpT is conserved in all vertebrates MARKs, we conclude that this sequence is crucial for MARK-dependent regulation of cellular polarity.

Klíčová slova:

293T cells – Cell membranes – Microtubules – Phosphorylation – Plasmid construction – Protein interactions – Sequence alignment – Sequence motif analysis


1. Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein kinase complement of the human genome. Science. 2002;298(5600):1912–34. Epub 2002/12/10. doi: 10.1126/science.1075762 12471243.

2. Shackelford DB, Shaw RJ. The LKB1-AMPK pathway: metabolism and growth control in tumour suppression. Nat Rev Cancer. 2009;9(8):563–75. Epub 2009/07/25. doi: 10.1038/nrc2676 19629071; PubMed Central PMCID: PMC2756045.

3. Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, Boudeau J, et al. LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. The EMBO journal. 2004;23(4):833–43. Epub 2004/02/21. doi: 10.1038/sj.emboj.7600110 14976552; PubMed Central PMCID: PMC381014.

4. Jaleel M, McBride A, Lizcano JM, Deak M, Toth R, Morrice NA, et al. Identification of the sucrose non-fermenting related kinase SNRK, as a novel LKB1 substrate. FEBS letters. 2005;579(6):1417–23. Epub 2005/03/01. doi: 10.1016/j.febslet.2005.01.042 15733851.

5. Jaleel M, Villa F, Deak M, Toth R, Prescott AR, Van Aalten DM, et al. The ubiquitin-associated domain of AMPK-related kinases regulates conformation and LKB1-mediated phosphorylation and activation. The Biochemical journal. 2006;394(Pt 3):545–55. Epub 2006/01/07. doi: 10.1042/BJ20051844 16396636; PubMed Central PMCID: PMC1383704.

6. Timm T, Li XY, Biernat J, Jiao J, Mandelkow E, Vandekerckhove J, et al. MARKK, a Ste20-like kinase, activates the polarity-inducing kinase MARK/PAR-1. The EMBO journal. 2003;22(19):5090–101. Epub 2003/10/01. doi: 10.1093/emboj/cdg447 14517247; PubMed Central PMCID: PMC204455.

7. Raman M, Earnest S, Zhang K, Zhao Y, Cobb MH. TAO kinases mediate activation of p38 in response to DNA damage. The EMBO journal. 2007;26(8):2005–14. Epub 2007/03/31. doi: 10.1038/sj.emboj.7601668 17396146; PubMed Central PMCID: PMC1852793.

8. Herzig S, Shaw RJ. AMPK: guardian of metabolism and mitochondrial homeostasis. Nature reviews Molecular cell biology. 2018;19(2):121–35. Epub 2017/10/05. doi: 10.1038/nrm.2017.95 28974774; PubMed Central PMCID: PMC5780224.

9. Chang S, Bezprozvannaya S, Li S, Olson EN. An expression screen reveals modulators of class II histone deacetylase phosphorylation. Proceedings of the National Academy of Sciences of the United States of America. 2005;102(23):8120–5. Epub 2005/06/01. doi: 10.1073/pnas.0503275102 15923258; PubMed Central PMCID: PMC1149448.

10. Sonntag T, Moresco JJ, Vaughan JM, Matsumura S, Yates JR 3rd, Montminy M. Analysis of a cAMP regulated coactivator family reveals an alternative phosphorylation motif for AMPK family members. PLoS One. 2017;12(2):e0173013. Epub 2017/02/25. doi: 10.1371/journal.pone.0173013 28235073.

11. Sonntag T, Vaughan JM, Montminy M. 14-3-3 proteins mediate inhibitory effects of cAMP on salt-inducible kinases (SIKs). The FEBS journal. 2018;285(3):467–80. Epub 2017/12/07. doi: 10.1111/febs.14351 29211348; PubMed Central PMCID: PMC5799007.

12. Walkinshaw DR, Weist R, Kim GW, You L, Xiao L, Nie J, et al. The tumor suppressor kinase LKB1 activates the downstream kinases SIK2 and SIK3 to stimulate nuclear export of class IIa histone deacetylases. The Journal of biological chemistry. 2013;288(13):9345–62. Epub 2013/02/09. doi: 10.1074/jbc.M113.456996 23393134; PubMed Central PMCID: PMC3611005.

13. Jansson D, Ng AC, Fu A, Depatie C, Al Azzabi M, Screaton RA. Glucose controls CREB activity in islet cells via regulated phosphorylation of TORC2. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(29):10161–6. Epub 2008/07/16. doi: 10.1073/pnas.0800796105 18626018; PubMed Central PMCID: PMC2481316.

14. Moravcevic K, Mendrola JM, Schmitz KR, Wang YH, Slochower D, Janmey PA, et al. Kinase associated-1 domains drive MARK/PAR1 kinases to membrane targets by binding acidic phospholipids. Cell. 2010;143(6):966–77. Epub 2010/12/15. doi: 10.1016/j.cell.2010.11.028 21145462; PubMed Central PMCID: PMC3031122.

15. Suzuki A, Hirata M, Kamimura K, Maniwa R, Yamanaka T, Mizuno K, et al. aPKC acts upstream of PAR-1b in both the establishment and maintenance of mammalian epithelial polarity. Current biology: CB. 2004;14(16):1425–35. Epub 2004/08/25. doi: 10.1016/j.cub.2004.08.021 15324659.

16. Goransson O, Deak M, Wullschleger S, Morrice NA, Prescott AR, Alessi DR. Regulation of the polarity kinases PAR-1/MARK by 14-3-3 interaction and phosphorylation. Journal of cell science. 2006;119(Pt 19):4059–70. Epub 2006/09/14. doi: 10.1242/jcs.03097 16968750.

17. Sakamoto K, Bultot L, Goransson O. The Salt-Inducible Kinases: Emerging Metabolic Regulators. Trends in endocrinology and metabolism: TEM. 2018;29(12):827–40. Epub 2018/11/06. doi: 10.1016/j.tem.2018.09.007 30385008.

18. Wu Y, Griffin EE. Regulation of Cell Polarity by PAR-1/MARK Kinase. Current topics in developmental biology. 2017;123:365–97. Epub 2017/02/27. doi: 10.1016/bs.ctdb.2016.11.001 28236972; PubMed Central PMCID: PMC5943083.

19. Drewes G, Ebneth A, Preuss U, Mandelkow EM, Mandelkow E. MARK, a novel family of protein kinases that phosphorylate microtubule-associated proteins and trigger microtubule disruption. Cell. 1997;89(2):297–308. Epub 1997/04/18. doi: 10.1016/s0092-8674(00)80208-1 9108484.

20. Wang Y, Mandelkow E. Tau in physiology and pathology. Nature reviews Neuroscience. 2016;17(1):5–21. Epub 2015/12/04. doi: 10.1038/nrn.2015.1 26631930.

21. Schwalbe M, Biernat J, Bibow S, Ozenne V, Jensen MR, Kadavath H, et al. Phosphorylation of human Tau protein by microtubule affinity-regulating kinase 2. Biochemistry. 2013;52(50):9068–79. Epub 2013/11/21. doi: 10.1021/bi401266n 24251416.

22. Hanger DP, Anderton BH, Noble W. Tau phosphorylation: the therapeutic challenge for neurodegenerative disease. Trends in molecular medicine. 2009;15(3):112–9. Epub 2009/02/28. doi: 10.1016/j.molmed.2009.01.003 19246243.

23. Simic G, Babic Leko M, Wray S, Harrington C, Delalle I, Jovanov-Milosevic N, et al. Tau Protein Hyperphosphorylation and Aggregation in Alzheimer's Disease and Other Tauopathies, and Possible Neuroprotective Strategies. Biomolecules. 2016;6(1):6. Epub 2016/01/12. doi: 10.3390/biom6010006 26751493; PubMed Central PMCID: PMC4808800.

24. Schneider A, Biernat J, von Bergen M, Mandelkow E, Mandelkow EM. Phosphorylation that detaches tau protein from microtubules (Ser262, Ser214) also protects it against aggregation into Alzheimer paired helical filaments. Biochemistry. 1999;38(12):3549–58. Epub 1999/03/26. doi: 10.1021/bi981874p 10090741.

25. Palmqvist S, Zetterberg H, Mattsson N, Johansson P, Minthon L, Blennow K, et al. Detailed comparison of amyloid PET and CSF biomarkers for identifying early Alzheimer disease. Neurology. 2015;85(14):1240–9. Epub 2015/09/12. doi: 10.1212/WNL.0000000000001991 26354982; PubMed Central PMCID: PMC4607601.

26. Ittner LM, Ke YD, Delerue F, Bi M, Gladbach A, van Eersel J, et al. Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer's disease mouse models. Cell. 2010;142(3):387–97. Epub 2010/07/27. doi: 10.1016/j.cell.2010.06.036 20655099.

27. Cummings J, Lee G, Ritter A, Zhong K. Alzheimer's disease drug development pipeline: 2018. Alzheimers Dement (N Y). 2018;4:195–214. Epub 2018/06/30. doi: 10.1016/j.trci.2018.03.009 29955663; PubMed Central PMCID: PMC6021548.

28. Lasagna-Reeves CA, de Haro M, Hao S, Park J, Rousseaux MW, Al-Ramahi I, et al. Reduction of Nuak1 Decreases Tau and Reverses Phenotypes in a Tauopathy Mouse Model. Neuron. 2016;92(2):407–18. Epub 2016/10/21. doi: 10.1016/j.neuron.2016.09.022 27720485; PubMed Central PMCID: PMC5745060.

29. Vingtdeux V, Davies P, Dickson DW, Marambaud P. AMPK is abnormally activated in tangle- and pre-tangle-bearing neurons in Alzheimer's disease and other tauopathies. Acta neuropathologica. 2011;121(3):337–49. Epub 2010/10/20. doi: 10.1007/s00401-010-0759-x 20957377; PubMed Central PMCID: PMC3060560.

30. Yoshida H, Goedert M. Phosphorylation of microtubule-associated protein tau by AMPK-related kinases. Journal of neurochemistry. 2012;120(1):165–76. Epub 2011/10/12. doi: 10.1111/j.1471-4159.2011.07523.x 21985311.

31. Mairet-Coello G, Courchet J, Pieraut S, Courchet V, Maximov A, Polleux F. The CAMKK2-AMPK kinase pathway mediates the synaptotoxic effects of Abeta oligomers through Tau phosphorylation. Neuron. 2013;78(1):94–108. Epub 2013/04/16. doi: 10.1016/j.neuron.2013.02.003 23583109; PubMed Central PMCID: PMC3784324.

32. Kishi M, Pan YA, Crump JG, Sanes JR. Mammalian SAD kinases are required for neuronal polarization. Science. 2005;307(5711):929–32. Epub 2005/02/12. doi: 10.1126/science.1107403 15705853.

33. Sonntag T, Ostojic J, Vaughan JM, Moresco JJ, Yoon YS, Yates JR, 3rd, et al. Mitogenic Signals Stimulate the CREB Coactivator CRTC3 through PP2A Recruitment. iScience. 2019;11:134–45. Epub 2019/01/06. doi: 10.1016/j.isci.2018.12.012 30611118; PubMed Central PMCID: PMC6317279.

34. Henriksson E, Jones HA, Patel K, Peggie M, Morrice N, Sakamoto K, et al. The AMPK-related kinase SIK2 is regulated by cAMP via phosphorylation at Ser358 in adipocytes. The Biochemical journal. 2012;444(3):503–14. Epub 2012/04/03. doi: 10.1042/BJ20111932 22462548; PubMed Central PMCID: PMC3631101.

35. Hirota T, Lee JW, St John PC, Sawa M, Iwaisako K, Noguchi T, et al. Identification of small molecule activators of cryptochrome. Science. 2012;337(6098):1094–7. Epub 2012/07/17. doi: 10.1126/science.1223710 22798407; PubMed Central PMCID: PMC3589997.

36. Sonntag T, Mootz HD. An intein-cassette integration approach used for the generation of a split TEV protease activated by conditional protein splicing. Mol Biosyst. 2011;7(6):2031–9. Epub 2011/04/14. doi: 10.1039/c1mb05025g 21487580.

37. Wolters DA, Washburn MP, Yates JR 3rd. An automated multidimensional protein identification technology for shotgun proteomics. Anal Chem. 2001;73(23):5683–90. Epub 2002/01/05. doi: 10.1021/ac010617e 11774908.

38. He L, Diedrich J, Chu YY, Yates JR 3rd. Extracting Accurate Precursor Information for Tandem Mass Spectra by RawConverter. Anal Chem. 2015;87(22):11361–7. Epub 2015/10/27. doi: 10.1021/acs.analchem.5b02721 26499134; PubMed Central PMCID: PMC4777630.

39. Peng J, Elias JE, Thoreen CC, Licklider LJ, Gygi SP. Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J Proteome Res. 2003;2(1):43–50. Epub 2003/03/20. doi: 10.1021/pr025556v 12643542.

40. Xu T, Park SK, Venable JD, Wohlschlegel JA, Diedrich JK, Cociorva D, et al. ProLuCID: An improved SEQUEST-like algorithm with enhanced sensitivity and specificity. J Proteomics. 2015;129:16–24. Epub 2015/07/15. doi: 10.1016/j.jprot.2015.07.001 26171723; PubMed Central PMCID: PMC4630125.

41. Tabb DL, McDonald WH, Yates JR 3rd. DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J Proteome Res. 2002;1(1):21–6. Epub 2003/03/20. doi: 10.1021/pr015504q 12643522; PubMed Central PMCID: PMC2811961.

42. The UniProt Consortium. UniProt: the universal protein knowledgebase. Nucleic Acids Res. 2017;45(D1):D158–D69. Epub 2016/12/03. doi: 10.1093/nar/gkw1099 27899622; PubMed Central PMCID: PMC5210571.

43. Nesic D, Miller MC, Quinkert ZT, Stein M, Chait BT, Stebbins CE. Helicobacter pylori CagA inhibits PAR1-MARK family kinases by mimicking host substrates. Nat Struct Mol Biol. 2010;17(1):130–2. Epub 2009/12/08. doi: 10.1038/nsmb.1705 19966800; PubMed Central PMCID: PMC3006182.

44. Marx A, Nugoor C, Panneerselvam S, Mandelkow E. Structure and function of polarity-inducing kinase family MARK/Par-1 within the branch of AMPK/Snf1-related kinases. FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 2010;24(6):1637–48. Epub 2010/01/15. doi: 10.1096/fj.09-148064 20071654.

45. Xu C, Jin J, Bian C, Lam R, Tian R, Weist R, et al. Sequence-specific recognition of a PxLPxI/L motif by an ankyrin repeat tumbler lock. Science signaling. 2012;5(226):ra39. Epub 2012/06/01. doi: 10.1126/scisignal.2002979 22649097.

46. Galjart N. CLIPs and CLASPs and cellular dynamics. Nature reviews Molecular cell biology. 2005;6(6):487–98. Epub 2005/06/02. doi: 10.1038/nrm1664 15928712.

47. Altarejos JY, Montminy M. CREB and the CRTC co-activators: sensors for hormonal and metabolic signals. Nature reviews Molecular cell biology. 2011;12(3):141–51. Epub 2011/02/25. doi: 10.1038/nrm3072 21346730; PubMed Central PMCID: PMC4324555.

48. Wein MN, Foretz M, Fisher DE, Xavier RJ, Kronenberg HM. Salt-Inducible Kinases: Physiology, Regulation by cAMP, and Therapeutic Potential. Trends in endocrinology and metabolism: TEM. 2018. Epub 2018/08/29. doi: 10.1016/j.tem.2018.08.004 30150136.

49. Conkright MD, Canettieri G, Screaton R, Guzman E, Miraglia L, Hogenesch JB, et al. TORCs: transducers of regulated CREB activity. Molecular cell. 2003;12(2):413–23. Epub 2003/10/11. doi: 10.1016/j.molcel.2003.08.013 14536081.

50. Timm T, Balusamy K, Li X, Biernat J, Mandelkow E, Mandelkow EM. Glycogen synthase kinase (GSK) 3beta directly phosphorylates Serine 212 in the regulatory loop and inhibits microtubule affinity-regulating kinase (MARK) 2. The Journal of biological chemistry. 2008;283(27):18873–82. Epub 2008/04/22. doi: 10.1074/jbc.M706596200 18424437.

51. Trinczek B, Brajenovic M, Ebneth A, Drewes G. MARK4 is a novel microtubule-associated proteins/microtubule affinity-regulating kinase that binds to the cellular microtubule network and to centrosomes. The Journal of biological chemistry. 2004;279(7):5915–23. Epub 2003/11/05. doi: 10.1074/jbc.M304528200 14594945.

52. Kumar S, Tepper K, Kaniyappan S, Biernat J, Wegmann S, Mandelkow EM, et al. Stages and conformations of the Tau repeat domain during aggregation and its effect on neuronal toxicity. The Journal of biological chemistry. 2014;289(29):20318–32. Epub 2014/05/16. doi: 10.1074/jbc.M114.554725 24825901; PubMed Central PMCID: PMC4106345.

53. Yaffe MB. How do 14-3-3 proteins work?—Gatekeeper phosphorylation and the molecular anvil hypothesis. FEBS letters. 2002;513(1):53–7. Epub 2002/03/26. doi: 10.1016/s0014-5793(01)03288-4 11911880.

54. Miller CJ, Turk BE. Homing in: Mechanisms of Substrate Targeting by Protein Kinases. Trends in biochemical sciences. 2018;43(5):380–94. Epub 2018/03/17. doi: 10.1016/j.tibs.2018.02.009 29544874; PubMed Central PMCID: PMC5923429.

55. Alfonso SI, Callender JA, Hooli B, Antal CE, Mullin K, Sherman MA, et al. Gain-of-function mutations in protein kinase Calpha (PKCalpha) may promote synaptic defects in Alzheimer's disease. Science signaling. 2016;9(427):ra47. Epub 2016/05/12. doi: 10.1126/scisignal.aaf6209 27165780; PubMed Central PMCID: PMC5154619.

56. Westwood I, Cheary DM, Baxter JE, Richards MW, van Montfort RL, Fry AM, et al. Insights into the conformational variability and regulation of human Nek2 kinase. Journal of molecular biology. 2009;386(2):476–85. Epub 2009/01/07. doi: 10.1016/j.jmb.2008.12.033 19124027; PubMed Central PMCID: PMC2741569.

57. Cheng Y, Zhang Y, McCammon JA. How does activation loop phosphorylation modulate catalytic activity in the cAMP-dependent protein kinase: a theoretical study. Protein science: a publication of the Protein Society. 2006;15(4):672–83. Epub 2006/03/09. doi: 10.1110/ps.051852306 16522793; PubMed Central PMCID: PMC2242471.

58. Sandi MJ, Marshall CB, Balan M, Coyaud E, Zhou M, Monson DM, et al. MARK3-mediated phosphorylation of ARHGEF2 couples microtubules to the actin cytoskeleton to establish cell polarity. Science signaling. 2017;10(503). Epub 2017/11/02. doi: 10.1126/scisignal.aan3286 29089450.

59. Kruse R, Krantz J, Barker N, Coletta RL, Rafikov R, Luo M, et al. Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein. Molecular & cellular proteomics: MCP. 2017;16(10):1718–35. Epub 2017/05/28. doi: 10.1074/mcp.RA117.000011 28550165; PubMed Central PMCID: PMC5629260.

60. Emptage RP, Lemmon MA, Ferguson KM. Molecular determinants of KA1 domain-mediated autoinhibition and phospholipid activation of MARK1 kinase. The Biochemical journal. 2017;474(3):385–98. Epub 2016/11/24. doi: 10.1042/BCJ20160792 27879374; PubMed Central PMCID: PMC5317272.

61. Emptage RP, Lemmon MA, Ferguson KM, Marmorstein R. Structural Basis for MARK1 Kinase Autoinhibition by Its KA1 Domain. Structure. 2018;26(8):1137–43 e3. Epub 2018/08/14. doi: 10.1016/j.str.2018.05.008 30099988; PubMed Central PMCID: PMC6092042.

Článek vyšel v časopise


2019 Číslo 12
Nejčtenější tento týden