The role of CKIP-1 in cell morphology depends on its interaction with actin-capping protein.
CKIP-1 is a pleckstrin homology domain-containing protein that induces alterations of the actin cytoskeleton and cell morphology when expressed in human osteosarcoma cells. CKIP-1 interacts with the heterodimeric actin-capping protein in cells, so we postulated that this interaction was responsible for the observed cytoskeletal and morphological effects of CKIP-1. To test this postulate, we used peptide "walking arrays" and alignments of CKIP-1 with CARMIL, another CP-binding protein, to identify Arg-155 and Arg-157 of CKIP-1 as residues potentially required for its interactions with CP. CKIP-1 mutants harboring Arg-155 and Arg-157 substitutions exhibited greatly decreased CP binding, while retaining wild-type localization, the ability to interact with protein kinase CK2, and self-association. To examine the phenotype associated with expression of these mutants, we generated tetracycline-inducible human osteosarcoma cells lines expressing R155E,R157E mutants of CKIP-1. Examination of these cell lines reveals that CKIP-1 R155E,R157E did not induce the distinct changes in cell morphology and the actin cytoskeleton that are characteristic of wild-type CKIP-1 demonstrating that the interaction between CKIP-1 and CP is required for these cellular effects.
Canton
David A
DA
Regulatory Biology and Functional Genomics Research Group, Siebens-Drake Medical Research Institute, Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Olsten
Mary Ellen K
ME
Niederstrasser
Hanspeter
H
Cooper
John A
JA
Litchfield
David W
DW
D000199:Actins / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D047390:Casein Kinase II / Q000378:metabolism; D045744:Cell Line, Tumor; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D012516:Osteosarcoma / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010590:Phalloidine / Q000494:pharmacology; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
D000199:Actins; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C408877:PLEKHO1 protein, human; D010455:Peptides; D010590:Phalloidine; D001120:Arginine; D047390:Casein Kinase II