Abstract
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.
Highlights
CKIP-14 was identified in a yeast two-hybrid screen for novel interaction partners of protein kinase CK2 (1)
It is possible that the interaction of CP with CKIP-1 at the plasma membrane inhibits binding of CP to the barbed ends of the actin filament leading to increased actin polymerization and changes in cellular morphology
Co-immunoprecipitation of Endogenous CP with Endogenous CKIP-1—We have previously shown that elevated expression of CKIP-1 induces alterations in cell morphology and the actin cytoskeleton
Summary
CKIP-14 was identified in a yeast two-hybrid screen for novel interaction partners of protein kinase CK2 (1). This targeting of CK2 is lost when the PH domain of CKIP-1 is replaced by a myristoylation recognition sequence, even though the CKIP-1 mutant still localizes to the plasma membrane These results suggest that CKIP-1 may function in an analogous manner to protein kinase A anchoring proteins, which target cAMP-dependent protein kinase A (3– 6). It is possible that the interaction of CP with CKIP-1 at the plasma membrane inhibits binding of CP to the barbed ends of the actin filament leading to increased actin polymerization and changes in cellular morphology. The effects of CKIP-1 on cell morphology may require interactions with CP to target CKIP-1 to the barbed ends of actin filaments To distinguish between these models, we used peptide “walking arrays” and alignments with the CP-binding protein, CARMIL (11), to identify Arg-155 and Arg-157 as residues of CKIP-1 potentially required for its binding with CP. To directly test whether changes in cell morphology and the actin cytoskeleton induced by CKIP-1 require its interactions with CP, we generated human osteosarcoma cell lines expressing CP-binding deficient mutants of CKIP-1 under the control of tetracycline
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