The Pleckstrin Homology Domain of CK2 Interacting Protein-1 Is Required for Interactions and Recruitment of Protein Kinase CK2 to the Plasma Membrane

ABSTRACT 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 β-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, CPα and CPβ, as novel CKIP-1 interaction partners. Interactions were confirmed by coimmunoprecipitation and by colocalization. Furthermore, we demonstrate that Ser9 of CPα is phosphorylated by protein kinase CK2 in vitro, that CPα is phosphorylated in vivo, and that treatment with a CK2-specific inhibitor results in a decrease in CPα 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.

Download Full-text

Subtype-specific regulation of equilibrative nucleoside transporters by protein kinase CK2

Biochemical Journal ◽

10.1042/bj20041571 ◽

2005 ◽

Vol 386 (2) ◽

pp. 281-289 ◽

Cited By ~ 21

Author(s):

Meaghan STOLK ◽

Elizabeth COOPER ◽

Greg VILK ◽

David W. LITCHFIELD ◽

James R. HAMMOND

Keyword(s):

Protein Kinase ◽

Protein Kinase Ck2 ◽

Nucleoside Transporters ◽

Nucleoside Transport ◽

Nucleoside Transporter ◽

Interacting Protein ◽

Human Osteosarcoma Cells ◽

Specific Regulation ◽

Membrane Lifetime ◽

Kinase Ck2

Two subtypes of equilibrative transporters, es (equilibrative inhibitor-sensitive) and ei (equilibrative inhibitor-insensitive), are responsible for the majority of nucleoside flux across mammalian cell membranes. Sequence analyses of the representative genes, ENT1 {equilibrative nucleoside transporter 1; also known as SLC29A1 [solute carrier family 29 (nucleoside transporters), member 1]} and ENT2 (SLC29A2), suggest that protein kinase CK2-mediated phosphorylation may be involved in the regulation of es- and ei-mediated nucleoside transport. We used human osteosarcoma cells transfected with catalytically active or inactive α′ and α subunits of CK2 to assess the effects of CK2 manipulation on nucleoside transport activity. Expression of inactive CK2α′ (decreased CK2α′ activity) increased the number of binding sites (∼1.5-fold) for the es-specific probe [3H]NBMPR ([3H]nitrobenzylthioinosine), and increased (∼1.8-fold) the Vmax for 2-chloro[3H]adenosine of the NBMPR-sensitive (es) nucleoside transporter. There was a concomitant decrease in the Vmax of the NBMPR-resistant (ei-mediated) uptake of 2-chloro[3H]adenosine. This inhibition of CK2α′ activity had no effect, however, on either the KD of [3H]NBMPR binding or the Km of 2-chloro[3H]adenosine uptake. Quantitative PCR showed a transient decrease in the expression of both hENT1 (human ENT1) and hENT2 mRNAs within 4–12 h of induction of the inactive CK2α′ subunit, but both transcripts had returned to control levels by 24 h. These data suggest that inhibition of CK2α′ reduced ei activity by attenuation of hENT2 transcription, while the increase in es/hENT1 activity was mediated by post-translational action of CK2. The observed modification in es activity was probably due to a CK2α′-mediated change in the phosphorylation state of the ENT1 protein, or an interacting protein, effecting an increase in the plasma membrane lifetime of the transport proteins.

Download Full-text

The plasma membrane translocation of diacylglycerol kinase δ1 is negatively regulated by conventional protein kinase C-dependent phosphorylation at Ser-22 and Ser-26 within the pleckstrin homology domain

Biochemical Journal ◽

10.1042/bj20040681 ◽

2004 ◽

Vol 382 (3) ◽

pp. 957-966 ◽

Cited By ~ 39

Author(s):

Shin-ichi IMAI ◽

Masahiro KAI ◽

Keiko YAMADA ◽

Hideo KANOH ◽

Fumio SAKANE

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Diacylglycerol Kinase ◽

Pleckstrin Homology Domain ◽

Ph Domain ◽

Pleckstrin Homology ◽

Bioactive Lipids ◽

Intact Cells ◽

Kinase C

DGK (diacylglycerol kinase) regulates the concentration of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKδ1 or its PH (pleckstrin homology) domain alone has been shown to be translocated to the plasma membrane from the cytoplasm in PMA-treated cells. In the present study, we identified Ser-22 and Ser-26 within the PH domain as the PMA- and epidermal-growth-factor-dependent phosphorylation sites of DGKδ1. Experiments in vitro and with intact cells suggested that the cPKC (conventional protein kinase C) phosphorylated these Ser residues directly. Puzzlingly, alanine/asparagine mutants at Ser-22 and Ser-26 of DGKδ1 and its PH domain are still persistently translocated by PMA treatment, suggesting that the PH domain phosphorylation is not responsible for the enzyme translocation and that the translocation was caused by a PMA-dependent, but cPKC-independent, process yet to be identified. Interestingly, the aspartate mutation, which mimics phosphoserine, at Ser-22 or Ser-26, inhibited the translocation of full-length DGKδ1 and the PH domain markedly, suggesting that the phosphorylation regulates negatively the enzyme translocation. Our results provide evidence of the phosphorylation of the DGKδ1 PH domain by cPKC, and suggest that the phosphorylation is involved in the control of subcellular localization of DGKδ1.

Download Full-text