The pleckstrin homology domain-containing protein CKIP-1 is involved in regulation of cell morphology and the actin cytoskeleton and interaction with actin capping protein.
CKIP-1 is a pleckstrin homology domain-containing protein that interacts with protein kinase CK2. To elucidate the functions of CKIP-1, we generated human osteosarcoma cell lines with tetracycline-regulated expression of Flag-CKIP-1. Flag-CKIP-1 expression resulted in distinct changes in cellular morphology. Therefore, we examined the actin profile by immunofluorescence, quantitative measurement of phalloidin binding, and immunoblot analysis. These studies demonstrate that Flag-CKIP-1 expression resulted in increases in F-actin staining and protein levels of beta-actin. To elucidate the mechanisms behind the observed phenotype, we utilized tandem affinity purification to isolate CKIP-1 interacting proteins. Mass spectrometry analysis led to the identification of the actin capping protein subunits, CPalpha and CPbeta, as novel CKIP-1 interaction partners. Interactions were confirmed by coimmunoprecipitation and by colocalization. Furthermore, we demonstrate that Ser9 of CPalpha is phosphorylated by protein kinase CK2 in vitro, that CPalpha is phosphorylated in vivo, and that treatment with a CK2-specific inhibitor results in a decrease in CPalpha phosphorylation. Finally, we demonstrate that CKIP-1 and CK2 inhibit the activity of actin capping protein at the barbed ends of actin filaments. Overall, our results are consistent with CKIP-1 playing a role in the regulation of the actin cytoskeleton through its interactions with actin capping protein.
Canton
David A
DA
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
Olsten
Mary Ellen K
ME
Kim
Kyoungtae
K
Doherty-Kirby
Amanda
A
Lajoie
Gilles
G
Cooper
John A
JA
Litchfield
David W
DW
D051344:Actin Capping Proteins; D008841:Actin Cytoskeleton / Q000378:metabolism*; D051339:Actin Depolymerizing Factors; D000199:Actins / Q000032:analysis / Q000378:metabolism*; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D047390:Casein Kinase II / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D045744:Cell Line, Tumor; D051345:Destrin; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D012516:Osteosarcoma; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D021122:Protein Subunits / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D012694:Serine / Q000378:metabolism
D051344:Actin Capping Proteins; D051339:Actin Depolymerizing Factors; D000199:Actins; D002352:Carrier Proteins; D051345:Destrin; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; C408877:PLEKHO1 protein, human; D021122:Protein Subunits; D034741:RNA, Small Interfering; D012694:Serine; D047390:Casein Kinase II