Phosphorylation-dependent localization of microtubule-associated protein MAP2c to the actin cytoskeleton.
Microtubule-associated protein 2 (MAP2) is a neuronal phosphoprotein that promotes net microtubule growth and actin cross-linking and bundling in vitro. Little is known about MAP2 regulation or its interaction with the cytoskeleton in vivo. Here we investigate the in vivo function of three specific sites of phosphorylation on MAP2. cAMP-dependent protein kinase activity disrupts the MAP2-microtubule interaction in living HeLa cells and promotes MAP2c localization to peripheral membrane ruffles enriched in actin. cAMP-dependent protein kinase phosphorylates serines within three KXGS motifs, one within each tubulin-binding repeat. These highly conserved motifs are also found in homologous proteins tau and MAP4. Phosphorylation at two of these sites was detected in brain tissue. Constitutive phosphorylation at these sites was mimicked by single, double, and triple mutations to glutamic acid. Biochemical and microscopy-based assays indicated that mutation of a single residue was adequate to disrupt the MAP2-microtubule interaction in HeLa cells. Double or triple point mutation promoted MAP2c localization to the actin cytoskeleton. Specific association between MAP2c and the actin cytoskeleton was demonstrated by retention of MAP2c-actin colocalization after detergent extraction. Specific phosphorylation states may enhance the interaction of MAP2 with the actin cytoskeleton, thereby providing a regulated mechanism for MAP2 function within distinct cytoskeletal domains.
Ozer
R S
RS
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Halpain
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S
D000199:Actins / Q000032:analysis / Q000378:metabolism*; D000595:Amino Acid Sequence; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
D000199:Actins; C489788:MAP2 protein, human; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases