Comprehensive proteomic analysis of human Par protein complexes reveals an interconnected protein network.
The polarization of eukaryotic cells is controlled by the concerted activities of asymmetrically localized proteins. The PAR proteins, first identified in Caenorhabditis elegans, are common regulators of cell polarity conserved from nematode and flies to man. However, little is known about the molecular mechanisms by which these proteins and protein complexes establish cell polarity in mammals. We have mapped multiprotein complexes formed around the putative human Par orthologs MARK4 (microtubule-associated protein/microtubule affinity-regulating kinase 4) (Par-1), Par-3, LKB1 (Par-4), 14-3-3zeta and eta (Par-5), Par-6a, -b, -c, and PKClambda (PKC3). We employed a proteomic approach comprising tandem affinity purification (TAP) of protein complexes from cultured cells and protein sequencing by tandem mass spectrometry. From these data we constructed a highly interconnected protein network consisting of three core complex "modules" formed around MARK4 (Par-1), Par-3.Par-6, and LKB1 (Par-4). The network confirms most previously reported interactions. In addition we identified more than 50 novel interactors, some of which, like the 14-3-3 phospho-protein scaffolds, occur in more than one distinct complex. We demonstrate that the complex formation between LKB1.Par-4, PAPK, and Mo25 results in the translocation of LKB1 from the nucleus to the cytoplasm and to tight junctions and show that the LKB1 complex may activate MARKs, which are known to introduce 14-3-3 binding sites into several substrates. Our findings suggest co-regulation and/or signaling events between the distinct Par complexes and provide a basis for further elucidation of the molecular mechanisms that govern cell polarity.
Brajenovic
Miro
M
Cellzome AG, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Joberty
Gerard
G
Küster
Bernhard
B
Bouwmeester
Tewis
T
Drewes
Gerard
G
D048948:14-3-3 Proteins; D000091162:AMP-Activated Protein Kinase Kinases; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D006801:Humans; D007527:Isoenzymes; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry; D020543:Proteome; D017386:Sequence Homology, Amino Acid; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry; D000099293:Protein Kinase C-lambda
D048948:14-3-3 Proteins; D000091162:AMP-Activated Protein Kinase Kinases; D029742:Caenorhabditis elegans Proteins; D018076:DNA, Complementary; D007527:Isoenzymes; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D020543:Proteome; D014446:Tyrosine 3-Monooxygenase; D000099293:Protein Kinase C-lambda; C106207:par-5 protein, C elegans; C512642:par-6 protein, C elegans; C483047:MARK4 protein, human; C097238:PAR-3 protein, C elegans; C109790:STK11 protein, human