MARK4 Is a Novel Microtubule-associated Proteins/Microtubule Affinity-regulating Kinase That Binds to the Cellular Microtubule Network and to Centrosomes

The MARK protein kinases were originally identified by their ability to phosphorylate a serine motif in the microtubule-binding domain of tau that is critical for microtubule binding. Here, we report the cloning and expression of a novel human paralog, MARK4, which shares 75% overall homology with MARK1-3 and is predominantly expressed in brain. Homology is most pronounced in the catalytic domain (90%), and MARK4 readily phosphorylates tau and the related microtubule-associated protein 2 (MAP2) and MAP4. In contrast to the three paralogs that all exhibit uniform cytoplasmic localization, MARK4 colocalizes with the centrosome and with microtubules in cultured cells. Overexpression of MARK4 causes thinning out of the microtubule network, concomitant with a reorganization of microtubules into bundles. In line with these findings, we show that a tandem affinity-purified MARK4 protein complex contains α-, β-, and γ-tubulin. In differentiated neuroblastoma cells, MARK4 is localized prominently at the tips of neurite-like processes. We suggest that although the four MARK/PAR-1 kinases might play multiple cellular roles in concert with different targets, MARK4 is likely to be directly involved in microtubule organization in neuronal cells and may contribute to the pathological phosphorylation of tau in Alzheimer's disease. The MARK protein kinases were originally identified by their ability to phosphorylate a serine motif in the microtubule-binding domain of tau that is critical for microtubule binding. Here, we report the cloning and expression of a novel human paralog, MARK4, which shares 75% overall homology with MARK1-3 and is predominantly expressed in brain. Homology is most pronounced in the catalytic domain (90%), and MARK4 readily phosphorylates tau and the related microtubule-associated protein 2 (MAP2) and MAP4. In contrast to the three paralogs that all exhibit uniform cytoplasmic localization, MARK4 colocalizes with the centrosome and with microtubules in cultured cells. Overexpression of MARK4 causes thinning out of the microtubule network, concomitant with a reorganization of microtubules into bundles. In line with these findings, we show that a tandem affinity-purified MARK4 protein complex contains α-, β-, and γ-tubulin. In differentiated neuroblastoma cells, MARK4 is localized prominently at the tips of neurite-like processes. We suggest that although the four MARK/PAR-1 kinases might play multiple cellular roles in concert with different targets, MARK4 is likely to be directly involved in microtubule organization in neuronal cells and may contribute to the pathological phosphorylation of tau in Alzheimer's disease. Microtubules and their associated proteins (MAPs) 1The abbreviations used are: MAPsmicrotubule-associated proteinsMTmicrotubuleMARKMAP/microtubule affinity-regulating kinaseMTBDmicrotubule-binding domainMTOCmicrotubule organizing centerHAhemagglutininGFPenhanced green fluorescent proteinCHOChinese hamster ovaryHEKhuman embryonic kidneyKA domainkinase-associated domainPipes1,4-piperazinediethanesulfonic acidTRITCtetramethylrhodamine isothiocyanateRACErapid amplification of cDNA ends. provide a dynamic network that is critical for chromosome segregation in mitosis and for the establishment of cellular polarity in interphase cells, enabling asymmetric cell division and morphogenesis (1Moritz M. Agard D.A. Curr. Opin. Struct. Biol. 2001; 11: 174-181Crossref PubMed Scopus (139) Google Scholar, 2Gundersen G.G. Nat. Rev. Mol. Cell Biol. 2002; 3: 296-304Crossref PubMed Scopus (143) Google Scholar, 3Desai A. Mitchison T.J. Annu. Rev. Cell Dev. Biol. 1997; 13: 83-117Crossref PubMed Scopus (2007) Google Scholar). During these processes, microtubules serve as tracks for regulated movement and positioning of organelles and other membrane microdomains. Such processes require a dynamic microtubule array and are regulated by motor proteins and structural MAPs (4Scales S.J. Pepperkok R. Kreis T.E. Cell. 1997; 90: 1137-1148Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 5Ebneth A. Godemann R. Stamer K. Illenberger S. Trinczek B. Mandelkow E. J. Cell Biol. 1998; 143: 777-794Crossref PubMed Scopus (678) Google Scholar, 6Akhmanova A. Hoogenraad C.C. Drabek K. Stepanova T. Dortland B. Verkerk T. Vermeulen W. Burgering B.M. De Zeeuw C.I. Grosveld F. Galjart N. Cell. 2001; 104: 923-935Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 7Cassimeris L. Spittle C. Int. Rev. Cytol. 2001; 210: 163-226Crossref PubMed Scopus (187) Google Scholar). An intriguing case of cellular polarization is the outgrowth of neurites in neuroblasts or neuroblastoma cells. This process is not completely understood; however, it has been shown to involve the axonal microtubule-stabilizing proteins tau and MAP1B (8Brugg B. Reddy D. Matus A. Neuroscience. 1993; 52: 489-496Crossref PubMed Scopus (135) Google Scholar, 9Caceres A. Potrebic S. Kosik K.S. J. Neurosci. 1991; 11: 1515-1523Crossref PubMed Google Scholar) and the dendritic protein MAP2 (10Caceres A. Mautino J. Kosik K.S. Neuron. 1992; 9: 607-618Abstract Full Text PDF PubMed Scopus (227) Google Scholar, 11Chang L. Jones Y. Ellisman M.H. Goldstein L.S. Karin M. Dev. Cell. 2003; 4: 521-533Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). The binding of tau and MAP2 to microtubules is controlled by phosphorylation in a spatial and temporal fashion (12Mandell J.W. Banker G.A. Perspect. Dev. Neurobiol. 1996; 4: 125-135PubMed Google Scholar). The purification of the activity that was most effective in phosphorylating the microtubule-binding domain of the MAPs tau, MAP2, and MAP4 led to the identification of three kinases that were termed MAP/microtubule affinity-regulating kinases (MARKs) (13Drewes G. Trinczek B. Illenberger S. Biernat J. Schmitt-Ulms G. Meyer H.E. Mandelkow E.M. Mandelkow E. J. Biol. Chem. 1995; 270: 7679-7688Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 14Illenberger S. Drewes G. Trinczek B. Biernat J. Meyer H.E. Olmsted J.B. Mandelkow E.M. Mandelkow E. J. Biol. Chem. 1996; 271: 10834-10843Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). MARKs phosphorylate tau and related MAPs on their tubulin binding repeats and consequently catalyze their detachment from microtubules in vitro and in cultured cells (13Drewes G. Trinczek B. Illenberger S. Biernat J. Schmitt-Ulms G. Meyer H.E. Mandelkow E.M. Mandelkow E. J. Biol. Chem. 1995; 270: 7679-7688Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 15Drewes G. Ebneth A. Preuss U. Mandelkow E.M. Mandelkow E. Cell. 1997; 89: 297-308Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar, 16Jenkins S.M. Johnson G.V. J. Neurochem. 2000; 74: 1463-1468Crossref PubMed Scopus (26) Google Scholar). Overexpression of high levels of MARKs in cells causes the disruption of the cellular microtubule network (15Drewes G. Ebneth A. Preuss U. Mandelkow E.M. Mandelkow E. Cell. 1997; 89: 297-308Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar, 17Ebneth A. Drewes G. Mandelkow E. Cell Motil. Cytoskeleton. 1999; 44: 209-224Crossref PubMed Scopus (131) Google Scholar). Intriguingly, the major serine in tau specifically phosphorylated by MARK, serine 262, is hyperphosphorylated in tau from the neurofibrillary deposits found in Alzheimer's disease brains (18Hasegawa M. Morishima-Kawashima M. Takio K. Suzuki M. Titani K. Ihara Y. J. Biol. Chem. 1992; 267: 17047-17054Abstract Full Text PDF PubMed Google Scholar). This finding is in agreement with the observation that tau purified from such neurofibrillary deposits has lost the ability to bind to microtubules, which can be restored by dephosphorylation (19Alonso A.C. Zaidi T. Grundke-Iqbal I. Iqbal K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5562-5566Crossref PubMed Scopus (620) Google Scholar). microtubule-associated proteins microtubule MAP/microtubule affinity-regulating kinase microtubule-binding domain microtubule organizing center hemagglutinin enhanced green fluorescent protein Chinese hamster ovary human embryonic kidney kinase-associated domain 1,4-piperazinediethanesulfonic acid tetramethylrhodamine isothiocyanate rapid amplification of cDNA ends. Candidate orthologs of the mammalian MARKs have been described in fission yeast (20Levin D.E. Bishop J.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8272-8276Crossref PubMed Scopus (79) Google Scholar), nematodes (21Guo S. Kemphues K.J. Cell. 1995; 81: 611-620Abstract Full Text PDF PubMed Scopus (900) Google Scholar), frogs (22Ossipova O. He X. Green J. Gene Expr. Patterns. 2002; 2: 145-150Crossref PubMed Scopus (5) Google Scholar), and flies (23Shulman J.M. Benton R. St Johnston D. Cell. 2000; 101: 377-388Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 24Tomancak P. Piano F. Riechmann V. Gunsalus K.C. Kemphues K.J. Ephrussi A. Nat. Cell Biol. 2000; 2: 458-460Crossref PubMed Scopus (145) Google Scholar). Interestingly, in these organisms, the MARK-like kinases are involved in processes related to the establishment of cell polarity. The MARK ortholog in Schizosaccharomyces pombe, kin1p, is necessary for proper growth initiation from the cell tips (25Drewes G. Nurse P. FEBS Lett. 2003; 554: 45-49Crossref PubMed Scopus (37) Google Scholar). In Caenorhabditis elegans, defective PAR-1 prevents the early asymmetric cell divisions of the germ line lineage (21Guo S. Kemphues K.J. Cell. 1995; 81: 611-620Abstract Full Text PDF PubMed Scopus (900) Google Scholar), and in Drosophila melanogaster, PAR-1 is necessary for microtubule-driven processes both early in the germ line for establishing cyst polarity and oocyte specification (26Huynh J.R. Shulman J.M. Benton R. St. Johnston D. Development. 2001; 128: 1201-1209PubMed Google Scholar, 27Cox D.N. Lu B. Sun T.Q. Williams L.T. Jan Y.N. Curr. Biol. 2001; 11: 75-87Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and later for the formation of the embryonic axe (23Shulman J.M. Benton R. St Johnston D. Cell. 2000; 101: 377-388Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 24Tomancak P. Piano F. Riechmann V. Gunsalus K.C. Kemphues K.J. Ephrussi A. Nat. Cell Biol. 2000; 2: 458-460Crossref PubMed Scopus (145) Google Scholar). Recently, it was reported that Drosophila PAR-1 also functions as a molecular switch by phosphorylating Dishevelled, thus enabling it to transduce the Wnt signal to β-catenin (28Sun T.Q. Lu B. Feng J.J. Reinhard C. Jan Y.N. Fantl W.J. Williams L.T. Nat. Cell Biol. 2001; 3: 628-636Crossref PubMed Scopus (212) Google Scholar), linking polarity regulation by MARK kinases to molecular pathways that involve β-catenin in cancer (29Kinzler K.W. Vogelstein B. Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4321) Google Scholar), neurodegeneration, and apoptosis (30Zhang Z. Hartmann H. Do V.M. Abramowski D. Sturchler-Pierrat C. Staufenbiel M. Sommer B. van de Wetering M. Clevers H. Saftig P. De Strooper B. He X. Yankner B.A. Nature. 1998; 395: 698-702Crossref PubMed Scopus (477) Google Scholar). The few studies performed in mammalian cells have supported the notion that the role of MARK/PAR-1 kinases in the maintenance and establishment of cell polarity is conserved. Overexpression of MARK2 abrogates epithelial polarity in Madin-Darby canine kidney cells (31Bohm H. Brinkmann V. Drab M. Henske A. Kurzchalia T.V. Curr. Biol. 1997; 7: 603-606Abstract Full Text Full Text PDF PubMed Google Scholar), and MARK1 depletion by an antisense probe in PC12 cells or the expression of a catalytically inactive mutant in neuroblastoma cells inhibits process outgrowth in these systems (32Brown A.J. Hutchings C. Burke J.F. Mayne L.V. Mol. Cell. Neurosci. 1999; 13: 119-130Crossref PubMed Scopus (26) Google Scholar, 33Biernat J. Wu Y.Z. Timm T. Zheng-Fischhofer Q. Mandelkow E. Meijer L. Mandelkow E.M. Mol. Biol. Cell. 2002; 13: 4013-4028Crossref PubMed Scopus (235) Google Scholar). A comprehensive sequence analysis of the protein kinases of the human genome (34Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6459) Google Scholar) shows that the human MARK/PAR-1 gene family consists of four paralogous genes, of which one has not yet been characterized. In this study, we describe the cloning and characterization of the MARK4 protein kinase and demonstrate that MARK4 is distinct from the other members of the MARK kinase family, in that it associates with microtubules, centrosomes, and neurite-like processes of neuroblastoma cells. cDNA Cloning—Cosmid R31237 from chromosome 19 contained part of the open reading frame of a novel MARK family member, which we termed MARK4, and was used for the design of primer 5′-GGCTCAGAGCTCGAGGTCGT TGGAGATG-3′ to amplify the complete open reading frame from human fetal brain Marathon cDNA (BD Biosciences, Clontech) by RACE-PCR using rTthXL polymerase (PerkinElmer Life Sciences) and the adaptor-specific primer AP1 (BD Clontech). For reasons of sequence fidelity, the 3′ portion of the amplified open reading frame was then replaced by the ScaI-XhoI fragment from an EST from human neuroepithelium, IMAGE:531830 (RZPD, Deutsches Ressourcenzentrum fuer Genomforschung, Berlin, Germany). The open reading frame was fully sequenced on both strands, and the sequence was deposited into GenBank™ (accession number AY057448). For expression, the MARK4 open reading frame was fused N-terminally with a HA tag (15Drewes G. Ebneth A. Preuss U. Mandelkow E.M. Mandelkow E. Cell. 1997; 89: 297-308Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar) or with GFP (BD Biosciences, Clontech) and subcloned into pZome1 (Cellzome AG, Heidelberg, Germany) or pcDNA3 (Invitrogen) via BamHI and KpnI restriction sites. Between the tag sequences and the ATG of the open reading frame, we inserted an oligonucleotide cassette encoding a tobacco etch virus protease cleavage site as described previously (35Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2302) Google Scholar). To test whether the expression vector itself or the site where GFP is fused to MARK4 has any effect on the expression and intracellular location of the fluorescent signal, MARK4 was also subcloned via Gateway™ cloning into pcDNA-DEST47 and pcDNA-DEST53 providing a C- or N-terminal GFP tag, respectively (Invitrogen). Human MARK3 was amplified by RT-PCR from human fetal brain mRNA (BD Clontech) using rTthXL polymerase and primers 5′-CAGGCGCCTCCTCCGCAGCC-3′ and 5′-ACCTGAGAAAAAGTGAATTTTTA-3′, cloned into pCDNA3, and sequenced on both strands, and the sequence was deposited into GenBank™ (accession number AF465413). Human MARK2 and γ-tubulin were subcloned by PCR from cDNA clones IMAGE:3850417 and IMAGE:3345973, respectively (RZPD) and subcloned into pCDNA3 and pZome1, respectively. HA-tagged rat MARK1 was expressed from a pCDNA3 plasmid as described previously (15Drewes G. Ebneth A. Preuss U. Mandelkow E.M. Mandelkow E. Cell. 1997; 89: 297-308Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar). A catalytically inactive mutant of MARK4 was generated by mutating residues threonine 214 and serine 218 both into Ala using the mutagenic primer pair 5′-TCGAAGCTGGACGCCTTCTGCGGGGCCCCCCCTTATGCCGCCCCG-3′ and 5′-CGGGGCGGCATAAGGGGGGGCCCCGCAGAAGGCGTCCAGCTTCGA-3′ and the QuikChange kit (Stratagene) according to the manufacturer's instructions. Cell Culture—Chinese hamster ovary (CHO-K1) cells were grown in Ham's F-12 medium and human embryonic kidney cells (HEK293, ATCC, Manassas, VA) were grown in Dulbecco's modified Eagle's medium (Invitrogen), both supplemented with 10% fetal calf serum and 2 mm glutamine (Biochrom, Berlin, Germany). For Neuro2a cells (ATCC), minimum Earle medium, 10% fetal calf serum, 2 mm glutamine was used. Differentiation of Neuro2a cells was induced for 24-48 h adding only 0.1% fetal calf serum to minimum Earle medium. For fluorescence investigation of fixed or living cells, cell lines were grown onto coverslips or seeded onto LabTek chambered cover glass (NUNC, Naperville, IL) at 37 °C and 5% CO2 in a humidified chamber. Plasmids (150 ng) were transfected either into CHO or Neuro2a cells at 70% confluency by lipofection using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. HEK293 cells were transfected by calcium-phosphate precipitation using 8 μg of DNA mixed with 62.5 μl of 2 m CaCl2, 2× Hanks'-buffered solution (280 mm NaCl, 10 mm KCl, 1.5 mm Na2HPO4, 12 mm glucose, 50 mm Hepes, pH 7) in a final volume of 0.5 ml, which was incubated for 30 min at room temperature and used for a 10-cm plate of cells at ∼30% confluency. Fluorescence Microscopy—For immunofluorescence analysis, cells were fixed either with methanol for 10 min at -20 °C (Fig. 6, A-C) or 2% (v/v) paraformaldehyde for 20 min at 37 °C (Fig. 3). After fixation, cells were blocked with 5% bovine serum albumin, 0.1% Triton X-100 in phosphate-buffered saline. Primary antibody incubation was performed in phosphate-buffered saline, 3% goat serum for 1 h at 37 °C. Monoclonal mouse anti-γ-tubulin antibody (Sigma) and polyclonal rabbit anti-GFP antibody (BD Biosciences Clontech) were used. Cells were washed in phosphate-buffered saline and incubated for 1 h at 37 °C with secondary antibodies (fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG and Cy3-conjugated donkey anti-mouse IgG, both from Jackson ImmunoResearch, West Grove, PA). For paraformaldehyde fixation, cells were washed in microtubule-stabilizing buffer (80 mm Pipes, pH 6.9, 4% (w/v) polyethylene glycol 6,000, 1 mm MgCl2, 1 mm EGTA) for 5 min at 37 °C, fixed for 20 min at 37 °C in 2% (w/v) paraformaldehyde in phosphate-buffered saline, and permeabilized in 0.2% Triton X-100. Antibody treatment was performed using polyclonal rabbit anti-HA antibody and monoclonal mouse anti-α-tubulin antibody followed by incubation with TRITC-conjugated anti-rabbit antibody and fluorescein isothiocyanate-conjugated anti-mouse antibody (Sigma). Coverslips were mounted in Permafluor (Dianova, Hamburg, Germany) and analyzed the following day. For life observation (Figs. 4 and 5), cells were directly examined 16-18 h after transfection. Images were captured with an Axiovert fluorescence microscope (Zeiss, Oberkochen, Germany) equipped with a ×63 oil immersion objective, a CCD-camera using filters optimized for double-labeled experiments, and with Photoshop software (Adobe Systems, San José, CA) implemented for image processing.Fig. 3MARK kinases exhibit distinct cytoplasmic localization patterns, but only MARK4 colocalizes with microtubules. HA tag plasmid alone (A) or HA tag containing different tagged MARK constructs as indicated were transiently expressed in CHO cells, and cells were stained with antibodies against the HA epitope (left panels) and against tubulin (right panels). B and C, MARK4 exhibits colocalization with microtubules and induces thinning of the microtubule array and rearrangement into bundles. D and E, MARK4-T214A/S218A, a catalytically inactive MARK4 point mutant, does not colocalize with microtubules or induce bundling. F-H, MARK1, MARK2, or MARK3 does not exhibit colocalization with microtubules. Scale bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4GFP-tagged MARK4 localizes to the MTOC in transfected CHO cells and induces microtubule bundling. Living CHO cells were transfected with GFP-MARK4 or the inactive mutant GFP-MARK4-T214A/S218A and analyzed by epifluorescent microscopy. A-C, representative cells expressing GFP-MARK4 at 6, 12, and 48 h after transfection. D-F, representative cells expressing GFP-MARK4-T214A/S218A at 6, 12, and 48 h after transfection. 6 h after transfection, both active and inactive MARK4 label the centrosome (A and D). 12 h after transfection, active but not inactive MARK4 labels microtubules originating from the MTOC (B and E). 48 h after transfection, most cells round up and die (C and F). The micrographs in G-L show examples of MARK4-labeled microtubules in detail. Two (three in J) centrosomes nearby the nucleus (N) and filamentous structures emanating from the centrosomes, which likely represent bundles of microtubules. The centrosomes become clearly visible even with epifluorescent microscopy because MARK4 decreases the number of microtubules within the cells by inducing bundling. In K, only six microtubules are attached to one centrosome, whereas the other shows no microtubules at all in the focal plane (arrow). Scale bar, 2 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5GFP-MARK4 labels microtubules and centrosomes in undifferentiated Neuro2A cells and tips of growing processes in differentiating cells.A-F, undifferentiated Neuro2a cells were transfected with GFP-MARK4 and visualized 24 h post-transfection. As a result of the decreased number of microtubules, the centrosomes become clearly visible (see arrows in E and F). The fluorescent labeling of the MTOC and microtubule filaments originating from it is similar to the signal observed in CHO cells (Fig. 4). Amplified centrosomes are also labeled by GFP-MARK4 (A and B). Scale bar, 4 μm. G-J, Neuro2a cells differentiated by serum starvation and visualized 16 h post-transfection, showing GFP labeling of microtubules in the cell body. For many cells, only faint labeling is observed along the elongated processes (middle), possibly because of the lack of microtubules (H and I). Notably, the signal always increases again at the tip of these processes (left, arrows). Scale bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Immunoprecipitation and Kinase Assays—48 h after transfection, HEK293 cells were collected from one 10-cm dish in 400 μl of lysis buffer (250 mm Tris pH 7.5, 25% glycerol, 7.5 mm MgCl2, 0.5% (v/v) Triton X-100, 500 mm NaCl, 125 mm NaF, 5 mm Na3VO4, 1 mm dithiothreitol, Roche protease inhibitor tablets). Lysates were cleared by centrifugation for 20 min at 14,000 × g at 4 °C. A 10-μl sample of the supernatant was removed for immunoblotting. The remainder was used for immunoprecipitation with anti-Myc (BD Biosciences Clontech) or anti-HA resin (Roche Diagnostics) for 2 h at 4 °C. Immunoprecipitates were washed three times with lysis buffer and once with lysis buffer without Triton X-100. Beads were resuspended in SDS-PAGE sample buffer and separated by SDS-PAGE. Proteins were transferred to nitrocellulose membrane and detected using either horseradish peroxidase-conjugated 3F10 at 1/20,000 for horseradish peroxidase-conjugated 9E10 at 1/10,000 (BD Biosciences Clontech). Tandem affinity purification of MARK4 was performed from HEK293 cells stably transduced with pZome1-MARK4, and copurifying proteins were sequenced by LC-MS/MS essentially as described previously (36Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (4040) Google Scholar). For kinase assays, immunoprecipitates were washed three times with lysis buffer as above and once with kinase buffer (50 mm Hepes, pH 7.4, 10 mm MgCl2, 200 μm ATP, 100 mm NaCl, 1 mm dithiothreitol). Beads were incubated with 10 μl of kinase buffer supplemented with 2 μCi of [γ-32P]ATP and 1 μg of protein substrate) for 30 min at 30 °C. To stop the reaction, 5 μl of boiling 4× SDS-PAGE sample buffer was then added. In vitro phosphorylation studies were performed with bacterially expressed substrate proteins: human tau isoforms, MAP2c, and the tau-MTBD and MAP4-MTBD fragments (comprising the repeats of the microtubule-binding domain) as described previously (14Illenberger S. Drewes G. Trinczek B. Biernat J. Meyer H.E. Olmsted J.B. Mandelkow E.M. Mandelkow E. J. Biol. Chem. 1996; 271: 10834-10843Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Others—Northern blots were performed using 2 μg of poly(A)+ mRNA from different tissues, fractionated by denaturing agarose gel electrophoresis, and transferred to nylon membranes (BD Biosciences Clontech). Oligonucleotides 5′-CCTGCCCGCCGGTCCCGGAC-3′ and 5′-GCAACTTGTGACCTCAGGTC-3′ (MWG Biotech, Munich, Germany) were mixed and labeled to 107 cpm/pmol using [γ-32P]ATP and T4 polynucleotide kinase (New England Biolabs, Dreieich, Germany) and hybridized using QuickHyb solution according to the manufacturer (Stratagene, Heidelberg, Germany). Western blotting was performed as described previously (17Ebneth A. Drewes G. Mandelkow E. Cell Motil. Cytoskeleton. 1999; 44: 209-224Crossref PubMed Scopus (131) Google Scholar). Cloning and Sequence Analysis—A search for sequences encoding additional members of the human MARK/PAR-1 protein kinase family yielded two cosmids from chromosome 19 with pronounced sequence homology to MARK1, MARK2 (15Drewes G. Ebneth A. Preuss U. Mandelkow E.M. Mandelkow E. Cell. 1997; 89: 297-308Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar), and MARK3 (37Ono T. Kawabe T. Sonta S. Okamoto T. Cytogenet. Cell Genet. 1997; 79: 101-102Crossref PubMed Scopus (12) Google Scholar), representing a fourth paralog, which we termed MARK4. Using 5′-RACE, we cloned the complete open reading frame from fetal brain mRNA. The encoded protein contains the characteristic sequence domains of the MARK/PAR-1 kinase family (Fig. 1A). The overall sequence homology within the MARK family is 55% with a higher degree of homology among MARK1, MARK2, and MARK3 (70%). The kinase domain at the N terminus of the protein exhibits the highest degree of homology, slightly over 90%. Notably, the LDT(Pi)FCGS(Pi)P motif containing the two activating phosphorylation sites in the regulatory loop is conserved (15Drewes G. Ebneth A. Preuss U. Mandelkow E.M. Mandelkow E. Cell. 1997; 89: 297-308Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar). The catalytic domain is followed by the small ubiquitin-associated domain (38Hofmann K. Bucher P. Trends Biochem. Sci. 1996; 21: 172-173Abstract Full Text PDF PubMed Scopus (356) Google Scholar), which is thought to mediate interaction with ubiquitin (39Bertolaet B.L. Clarke D.J. Wolff M. Watson M.H. Henze M. Divita G. Reed S.I. Nat. Struct. Biol. 2001; 8: 417-422Crossref PubMed Scopus (277) Google Scholar). Adjacent to the ubiquitin-associated domain is a large less conserved “spacer” region and a conserved C-terminal kinase-associated (KA) domain (38Hofmann K. Bucher P. Trends Biochem. Sci. 1996; 21: 172-173Abstract Full Text PDF PubMed Scopus (356) Google Scholar), which is an exclusive feature of this protein family (Fig. 1A). In MARK4, the large spacer region is the least conserved, the overall homology with MARK1, MARK2, and MARK3 being <30%, thus setting MARK4 apart from MARK1-3 in a phylogenetic diagram (Fig. 1B). The human MARK4 locus is 19q13.3, which is close to a known susceptibility locus for Alzheimer's disease ascribed to the ApoE gene. The 4.4-kb MARK4 mRNA is ubiquitously expressed as analyzed by Northern blot with the highest levels in brain and testis. In testis, a larger mRNA, which may represent a splice variant, is also detected (Fig. 1C). MARK4 Phosphorylates tau Family MAPs on Their Microtubule-binding Domain—Given the extensive homology of the catalytic domain, we asked whether MARK4 specifically phosphorylates tau and related MAPs similar to MARK1 and MARK2 (15Drewes G. Ebneth A. Preuss U. Mandelkow E.M. Mandelkow E. Cell. 1997; 89: 297-308Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar). We fused the MARK4 open reading frame to an N-terminal HA tag and immunoprecipitated the fusion protein from transfected HEK293 cells. It readily phosphorylated recombinant tau (Fig. 2 B), a fragment of tau comprising only the microtubule-binding domain, and a synthetic peptide, TR1 (NVKSKIGSTENLK) derived from the first repeat of the tau microtubule-binding domain. When a 10-fold excess of the TR1 peptide was added in addition to full-length tau, the phosphorylation of tau was almost completely inhibited, indicating that the immunoprecipitated activity is specific (Fig. 2, A and B, lanes 1 and 2). After phosphorylation, the tau protein was detected by the phospho-tau antibody 12E8 (Fig. 2C), which recognizes tau phosphorylated at serine residues 262 and/or 356, the first and fourth KXGS motif within the tubulin-binding domain (40Seubert P. Mawal-Dewan M. Barbour R. Jakes R. Goedert M. Johnson G.V. Litersky J.M. Schenk D. Lieberburg I. Trojanowski J.Q. Lee V.M.-Y. J. Biol. Chem. 1995; 270: 18917-18922Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). As described previously for MARK1 and MARK2 (14Illenberger S. Drewes G. Trinczek B. Biernat J. Meyer H.E. Olmsted J.B. Mandelkow E.M. Mandelkow E. J. Biol. Chem. 1996; 271: 10834-10843Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), MARK4 also readily phosphorylates the related MAP2 and MAP4 (Fig. 2, A-C). In this in vitro phosphorylation assay, there was no significant difference betw