MELK (maternal embryonic leucine zipper kinase), which is a member of the AMPK (AMP-activated protein kinase)-related kinase family, plays important roles in diverse cellular processes and has become a promising drug target for certain cancers. However, the regulatory mechanism of MELK remains elusive. Here, we report the crystal structure of a fragment of human MELK that contains the kinase domain and ubiquitin-associated (UBA) domain. The UBA domain tightly binds to the back of the kinase domain, which may contribute to the proper conformation and activity of the kinase domain. Interestingly, the activation segment in the kinase domain displays a unique conformation that contains an intramolecular disulfide bond. The structural and biochemical analyses unravel the molecular mechanisms for the autophosphorylation/activation of MELK and the dependence of its catalytic activity on reducing agents. Thus, our results may provide the basis for designing specific MELK inhibitors for cancer treatment.
References
[1]
Heyer BS, Warsowe J, Solter D, Knowles BB, Ackerman SL (1997) New member of the Snf1/AMPK kinase family, Melk, is expressed in the mouse egg and preimplantation embryo. Mol Reprod Dev 47: 148–156.
[2]
Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, et al. (2004) LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J 23: 833–843.
[3]
Paris J, Philippe M (1990) Poly(A) metabolism and polysomal recruitment of maternal mRNAs during early Xenopus development. Dev Biol 140: 221–224.
[4]
Gil M, Yang Y, Lee Y, Choi I, Ha H (1997) Cloning and expression of a cDNA encoding a novel protein serine/threonine kinase predominantly expressed in hematopoietic cells. Gene 195: 295–301.
[5]
Mirey G, Chartrain I, Froment C, Quaranta M, Bouche JP, et al. (2005) CDC25B phosphorylated by pEg3 localizes to the centrosome and the spindle poles at mitosis. Cell Cycle 4: 806–811.
[6]
Seong HA, Gil M, Kim KT, Kim SJ, Ha H (2002) Phosphorylation of a novel zinc-finger-like protein, ZPR9, by murine protein serine/threonine kinase 38 (MPK38). Biochem J 361: 597–604.
[7]
Cordes S, Frank CA, Garriga G (2006) The C. elegans MELK ortholog PIG-1 regulates cell size asymmetry and daughter cell fate in asymmetric neuroblast divisions. Development 133: 2747–2756.
[8]
Davezac N, Baldin V, Blot J, Ducommun B, Tassan JP (2002) Human pEg3 kinase associates with and phosphorylates CDC25B phosphatase: a potential role for pEg3 in cell cycle regulation. Oncogene 21: 7630–7641.
[9]
Vulsteke V, Beullens M, Boudrez A, Keppens S, Van Eynde A, et al. (2004) Inhibition of spliceosome assembly by the cell cycle-regulated protein kinase MELK and involvement of splicing factor NIPP1. J Biol Chem 279: 8642–8647.
[10]
Saito R, Tabata Y, Muto A, Arai K, Watanabe S (2005) Melk-like kinase plays a role in hematopoiesis in the zebra fish. Mol Cell Biol 25: 6682–6693.
[11]
Nakano I, Paucar AA, Bajpai R, Dougherty JD, Zewail A, et al. (2005) Maternal embryonic leucine zipper kinase (MELK) regulates multipotent neural progenitor proliferation. J Cell Biol 170: 413–427.
[12]
Lin ML, Park JH, Nishidate T, Nakamura Y, Katagiri T (2007) Involvement of maternal embryonic leucine zipper kinase (MELK) in mammary carcinogenesis through interaction with Bcl-G, a pro-apoptotic member of the Bcl-2 family. Breast Cancer Res 9: R17.
[13]
Jung H, Seong HA, Ha H (2008) Murine protein serine/threonine kinase 38 activates apoptosis signal-regulating kinase 1 via Thr 838 phosphorylation. J Biol Chem 283: 34541–34553.
[14]
Gray D, Jubb AM, Hogue D, Dowd P, Kljavin N, et al. (2005) Maternal embryonic leucine zipper kinase/murine protein serine-threonine kinase 38 is a promising therapeutic target for multiple cancers. Cancer Res 65: 9751–9761.
[15]
Hebbard LW, Maurer J, Miller A, Lesperance J, Hassell J, et al. (2010) Maternal embryonic leucine zipper kinase is upregulated and required in mammary tumor-initiating cells in vivo. Cancer Res 70: 8863–8873.
[16]
Nakano I, Masterman-Smith M, Saigusa K, Paucar AA, Horvath S, et al. (2008) Maternal embryonic leucine zipper kinase is a key regulator of the proliferation of malignant brain tumors, including brain tumor stem cells. J Neurosci Res 86: 48–60.
[17]
Marie SK, Okamoto OK, Uno M, Hasegawa AP, Oba-Shinjo SM, et al. (2008) Maternal embryonic leucine zipper kinase transcript abundance correlates with malignancy grade in human astrocytomas. Int J Cancer 122: 807–815.
[18]
Pickard MR, Green AR, Ellis IO, Caldas C, Hedge VL, et al. (2009) Dysregulated expression of Fau and MELK is associated with poor prognosis in breast cancer. Breast Cancer Res 11: R60.
[19]
Nagase T, Seki N, Ishikawa K, Tanaka A, Nomura N (1996) Prediction of the coding sequences of unidentified human genes. V. The coding sequences of 40 new genes (KIAA0161-KIAA0200) deduced by analysis of cDNA clones from human cell line KG-1. DNA Res 3: 17–24.
[20]
Beullens M, Vancauwenbergh S, Morrice N, Derua R, Ceulemans H, et al. (2005) Substrate specificity and activity regulation of protein kinase MELK. J Biol Chem 280: 40003–40011.
[21]
Moravcevic K, Mendrola JM, Schmitz KR, Wang YH, Slochower D, et al. (2010) Kinase associated-1 domains drive MARK/PAR1 kinases to membrane targets by binding acidic phospholipids. Cell 143: 966–977.
[22]
Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinase complement of the human genome. Science 298: 1912–1934.
[23]
Madura K (2002) The ubiquitin-associated (UBA) domain: on the path from prudence to prurience. Cell Cycle 1: 235–244.
[24]
Jaleel M, Villa F, Deak M, Toth R, Prescott AR, et al. (2006) The ubiquitin-associated domain of AMPK-related kinases regulates conformation and LKB1-mediated phosphorylation and activation. Biochem J 394: 545–555.
[25]
Murphy JM, Korzhnev DM, Ceccarelli DF, Briant DJ, Zarrine-Afsar A, et al. (2007) Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain. Proc Natl Acad Sci U S A 104: 14336–14341.
[26]
Drewes G, Ebneth A, Preuss U, Mandelkow EM, Mandelkow E (1997) MARK, a novel family of protein kinases that phosphorylate microtubule-associated proteins and trigger microtubule disruption. Cell 89: 297–308.
[27]
Marx A, Nugoor C, Muller J, Panneerselvam S, Timm T, et al. (2006) Structural variations in the catalytic and ubiquitin-associated domains of microtubule-associated protein/microtubule affinity regulating kinase (MARK) 1 and MARK2. J Biol Chem 281: 27586–27599.
[28]
Hardie DG, Ross FA, Hawley SA (2012) AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat Rev Mol Cell Biol 13: 251–262.
[29]
Chen L, Jiao ZH, Zheng LS, Zhang YY, Xie ST, et al. (2009) Structural insight into the autoinhibition mechanism of AMP-activated protein kinase. Nature 459: 1146–1149.
[30]
Crute BE, Seefeld K, Gamble J, Kemp BE, Witters LA (1998) Functional domains of the alpha1 catalytic subunit of the AMP-activated protein kinase. J Biol Chem 273: 35347–35354.
[31]
Seong HA, Jung H, Ha H (2010) Murine protein serine/threonine kinase 38 stimulates TGF-beta signaling in a kinase-dependent manner via direct phosphorylation of Smad proteins. J Biol Chem 285: 30959–30970.
[32]
Seong HA, Ha H (2012) Murine Protein Serine-threonine Kinase 38 Activates p53 Function through Ser15 Phosphorylation. J Biol Chem 287: 20797–20810.
[33]
Seong HA, Jung H, Manoharan R, Ha H (2012) PDK1 Protein Phosphorylation at Thr354 by Murine Protein Serine-Threonine Kinase 38 Contributes to Negative Regulation of PDK1 Protein Activity. J Biol Chem 287: 20811–20822.
[34]
Dale S, Wilson WA, Edelman AM, Hardie DG (1995) Similar substrate recognition motifs for mammalian AMP-activated protein kinase, higher plant HMG-CoA reductase kinase-A, yeast SNF1, and mammalian calmodulin-dependent protein kinase I. FEBS Lett 361: 191–195.
[35]
McClure WR (1969) A kinetic analysis of coupled enzyme assays. Biochemistry 8: 2782–2786.
[36]
Roskoski R Jr (1983) Assays of protein kinase. Methods Enzymol 99: 3–6.
[37]
Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276: 307–326.
Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, et al. (2002) PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr 58: 1948–1954.
[40]
Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126–2132.
[41]
Panneerselvam S, Marx A, Mandelkow EM, Mandelkow E (2006) Structure of the catalytic and ubiquitin-associated domains of the protein kinase MARK/Par-1. Structure 14: 173–183.
[42]
Nesic D, Miller MC, Quinkert ZT, Stein M, Chait BT, et al. (2010) Helicobacter pylori CagA inhibits PAR1-MARK family kinases by mimicking host substrates. Nat Struct Mol Biol 17: 130–132.
[43]
Marx A, Nugoor C, Panneerselvam S, Mandelkow E (2010) Structure and function of polarity-inducing kinase family MARK/Par-1 within the branch of AMPK/Snf1-related kinases. Faseb J 24: 1637–1648.
[44]
Huse M, Kuriyan J (2002) The conformational plasticity of protein kinases. Cell 109: 275–282.
[45]
Nolen B, Taylor S, Ghosh G (2004) Regulation of protein kinases; controlling activity through activation segment conformation. Mol Cell 15: 661–675.
[46]
Kornev AP, Haste NM, Taylor SS, Eyck LF (2006) Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism. Proc Natl Acad Sci U S A 103: 17783–17788.
[47]
Kornev AP, Taylor SS, Ten Eyck LF (2008) A helix scaffold for the assembly of active protein kinases. Proc Natl Acad Sci U S A 105: 14377–14382.
[48]
Taylor SS, Kornev AP (2011) Protein kinases: evolution of dynamic regulatory proteins. Trends Biochem Sci 36: 65–77.
