Zasp/Cypher internal ZM-motif containing fragments are sufficient to co-localize with alpha-actinin--analysis of patient mutations.
Z-band alternatively spliced PDZ-containing protein (ZASP/Cypher) has an important role in maintaining Z-disc stability in striated and cardiac muscle. ZASP/Cypher interacts through its PDZ domain with the major Z-disc actin cross-linker, alpha-actinin. ZASP/Cypher also has a conserved sequence called the ZM-motif, and it is found in two alternatively spliced exons 4 and 6. We have shown earlier that the ZM-motif containing internal regions of two related proteins ALP and CLP36 interact with alpha-actinin rod region, and that the ZM-motif is important in targeting ALP to the alpha-actinin containing structures in cell. Here, we show that the ZASP/Cypher internal fragments containing either ZM exon 4 or 6 co-localized with alpha-actinin in cultured myoblasts and nonmuscle cells. Fragments of 130 residues around the ZM-consensus were sufficient for localization, which is similar to our previous results of ALP. Moreover, ZASP/Cypher protein interacted directly with the alpha-actinin rod and competed with ALP in binding to the rod. During the inhibition of stress fiber assembly ZASP/Cypher and alpha-actinin co-localization could be partially disturbed, suggesting that ZASP/Cypher is bound to alpha-actinin mainly when alpha-actinin is localizing in stress fibers. Many point mutations found in cardiomyopathy patients are located in the internal region of ZASP/Cypher. However, we found no evidence that human patient mutations in the internal domain would affect the ZASP/Cypher co-localization with alpha-actinin, or that the mutations would destabilize the ZASP/Cypher protein.
Biocenter Oulu and Department of Biochemistry, University of Oulu, PO Box 3000, FIN-90014 Oulu, Finland. tuula.klaavuniemi@oulu.fi
Tuula
T
Klaavuniemi
Jari
J
Ylänne
D000185:Actinin / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D016466:CHO Cells; D009202:Cardiomyopathies / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D006224:Cricetinae; D005091:Exons / Q000502:physiology; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D060588:LIM Domain Proteins; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000648:ultrastructure; D009154:Mutation / Q000235:genetics*; D032446:Myoblasts; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D015388:Organelles / Q000378:metabolism / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D012518:Sarcomeres / Q000378:metabolism / Q000648:ultrastructure; D022502:Stress Fibers / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C120696:LDB3 protein, human; D060588:LIM Domain Proteins; D010446:Peptide Fragments; D000185:Actinin