Obscurin targets ankyrin-B and protein phosphatase 2A to the cardiac M-line.
Ankyrin-B targets ion channels and transporters in excitable cells. Dysfunction in ankyrin-B-based pathways results in defects in cardiac physiology. Despite a wealth of knowledge regarding the role of ankyrin-B for cardiac function, little is known regarding the mechanisms underlying ankyrin-B regulation. Moreover, the pathways underlying ankyrin-B targeting in heart are unclear. We report that alternative splicing regulates ankyrin-B localization and function in cardiomyocytes. Specifically, we identify a novel exon (exon 43') in the ankyrin-B regulatory domain that mediates interaction with the Rho-GEF obscurin. Ankyrin-B transcripts harboring exon 43' represent the primary cardiac isoform in human and mouse. We demonstrate that ankyrin-B and obscurin are co-localized at the M-line of myocytes and co-immunoprecipitate from heart. We define the structural requirements for ankyrin-B/obscurin interaction to two motifs in the ankyrin-B regulatory domain and demonstrate that both are critical for obscurin/ankyrin-B interaction. In addition, we demonstrate that interaction with obscurin is required for ankyrin-B M-line targeting. Specifically, both obscurin-binding motifs are required for the M-line targeting of a GFP-ankyrin-B regulatory domain. Moreover, this construct acts as a dominant-negative by competing with endogenous ankyrin-B for obscurin-binding at the M-line, thus providing a powerful new tool to evaluate the function of obscurin/ankyrin-B interactions. With this new tool, we demonstrate that the obscurin/ankyrin-B interaction is critical for recruitment of PP2A to the cardiac M-line. Together, these data provide the first evidence for the molecular basis of ankyrin-B and PP2A targeting and function at the cardiac M-line. Finally, we report that ankyrin-B R1788W is localized adjacent to the ankyrin-B obscurin-binding motif and increases binding activity for obscurin. In summary, our new findings demonstrate that ANK2 is subject to alternative splicing that gives rise to unique polypeptides with diverse roles in cardiac function.
Cunha
Shane R
SR
Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, USA. shane-cunha@uiowa.edu
Mohler
Peter J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005091:Exons / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009206:Myocardium / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases; D022821:RNA Splice Sites / Q000235:genetics; D064067:Rho Guanine Nucleotide Exchange Factors; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
C498994:ANK2 protein, human; C498995:Ank2 protein, mouse; D017487:Ankyrins; D020662:Guanine Nucleotide Exchange Factors; D009124:Muscle Proteins; D020033:Protein Isoforms; D022821:RNA Splice Sites; D064067:Rho Guanine Nucleotide Exchange Factors; D017346:Protein Serine-Threonine Kinases; C515766:obscn protein, mouse; D054648:Protein Phosphatase 2
J Biol Chem. 2004 Jun 11;279(24):25798-804
15075330
Am J Physiol Cell Physiol. 2004 Jul;287(1):C209-17
15013951
J Biol Chem. 2004 Sep 17;279(38):40185-93
15262991
J Biol Chem. 1993 May 5;268(13):9533-40
8486643
J Cell Biol. 2004 Sep 27;166(7):983-90
15381686
J Biol Chem. 1994 Nov 4;269(44):27163-6
7961622
J Biol Chem. 2004 Mar 26;279(13):12980-7
14722080
Cell. 2000 May 12;101(4):365-76
10830164
J Cell Biol. 1998 Sep 21;142(6):1571-81
9744885
J Cell Biol. 2003 Jan 20;160(2):245-53
12527750
J Cell Biol. 1996 Mar;132(5):835-48
8603916
J Neurosci. 1993 Sep;13(9):3725-35
8366343
Mol Biol Evol. 2006 Jan;23(1):46-55
16135777
J Neurosci. 2004 Aug 18;24(33):7230-40
15317849
J Cell Biol. 1991 Sep;114(6):1233-41
1716634
Exp Cell Res. 2005 Sep 10;309(1):86-98
15953600
J Mol Cell Cardiol. 1997 Jan;29(1):265-72
9040041
J Biol Chem. 2003 Feb 7;278(6):3985-91
12444090
Circulation. 2007 Jan 30;115(4):432-41
17242276
Science. 2005 Jun 10;308(5728):1599-603
15802564
J Biol Chem. 2007 Jan 19;282(3):2029-37
17074766
J Biol Chem. 1987 Aug 5;262(22):10537-45
3038887
Am J Physiol Heart Circ Physiol. 2000 Sep;279(3):H1307-18
10993798
Histochem Cell Biol. 2006 Mar;125(3):227-38
16205939
J Cell Biol. 2007 Jun 4;177(5):857-70
17548513
Mol Biol Cell. 2003 Mar;14(3):1138-48
12631729
J Cell Biol. 1996 May;133(4):819-30
8666667
Eur J Cell Biol. 1987 Jun;43(3):479-86
2957198
FASEB J. 2006 Oct;20(12):2102-11
17012262
J Neurosci. 2006 May 10;26(19):5230-9
16687515
Proc Natl Acad Sci U S A. 2004 Dec 14;101(50):17533-8
15579534
Curr Biol. 2001 Nov 27;11(23):1864-9
11728309
Nucleic Acids Res. 2001 Jul 1;29(13):E68-8
11433044
Nature. 1998 Oct 29;395(6705):863-9
9804419
J Cell Biol. 2006 Oct 23;175(2):325-35
17060500
J Biol Chem. 2006 Mar 3;281(9):5741-9
16368689
J Cell Biol. 2006 May 22;173(4):559-70
16702235
J Biol Chem. 1998 Jan 16;273(3):1339-48
9430667
J Biol Chem. 2007 Feb 16;282(7):4875-4883
17178715
Dev Biol. 2006 May 1;293(1):1-12
16566914
J Muscle Res Cell Motil. 2005;26(6-8):427-34
16625316
J Biol Chem. 2000 Dec 15;275(50):39710-7
10984483
J Cell Sci. 1997 May;110 ( Pt 10):1239-49
9191047
Nature. 1990 Mar 1;344(6261):36-42
2137557
J Cell Sci. 2006 Oct 15;119(Pt 20):4322-31
17038546
Science. 2001 Jul 6;293(5527):98-101
11441182
Nat Rev Genet. 2002 Apr;3(4):285-98
11967553
FEBS Lett. 2007 Apr 17;581(8):1549-54
17382936
Proc Natl Acad Sci U S A. 2004 Jun 15;101(24):9137-42
15178757
J Membr Biol. 2006;211(3):151-61
17091212
Exp Cell Res. 2006 Nov 1;312(18):3546-58
16962094
Physiol Rev. 1993 Oct;73(4):673-99
8415923
J Biol Chem. 1992 Sep 15;267(26):18966-72
1388161
J Biol Chem. 2002 Apr 12;277(15):12978-87
11796721
PLoS Biol. 2005 Dec;3(12):e423
16292983
Dev Dyn. 2006 Aug;235(8):2018-29
16779859
J Cell Biol. 1998 Nov 30;143(5):1305-15
9832558
J Cell Biol. 1997 Feb 10;136(3):621-31
9024692
J Cell Biol. 2008 Jan 14;180(1):173-86
18180363
Biochemistry. 2006 Mar 14;45(10):3448-59
16519540
J Biol Chem. 2004 Sep 24;279(39):40827-34
15247211
Nature. 2003 Feb 6;421(6923):634-9
12571597
J Biol Chem. 2003 Feb 21;278(8):6059-65
12464612
J Histochem Cytochem. 2004 Sep;52(9):1117-27
15314079
Proc Natl Acad Sci U S A. 1990 Mar;87(5):1730-4
1689849
Am J Physiol Heart Circ Physiol. 2007 Jul;293(1):H109-19
17416611
J Biol Chem. 2002 Mar 22;277(12):10599-607
11781319
Genomics. 1998 May 15;50(1):79-88
9628825
Circulation. 2007 Feb 13;115(6):743-51
17261657
Science. 2002 Jan 18;295(5554):496-9
11799244
J Biol Chem. 2007 Nov 2;282(44):32384-96
17720975
Heart Rhythm. 2005 Nov;2(11):1218-23
16253912
J Cell Biol. 2001 Jul 9;154(1):123-36
11448995