PMID- 15590896
OWN - NLM
STAT- MEDLINE
DCOM- 20050419
LR  - 20200930
IS  - 0363-6143 (Print)
IS  - 0363-6143 (Linking)
VI  - 288
IP  - 4
DP  - 2005 Apr
TI  - Involvement of NH2 terminus of PKC-delta in binding to F-actin during activation 
      of Calu-3 airway epithelial NKCC1.
PG  - C906-12
AB  - Direct binding of nonmuscle F-actin and the C2-like domain of PKC-delta 
      (deltaC2-like domain) is involved in hormone-mediated activation of epithelial 
      Na-K-2Cl cotransporter isoform 1 (NKCC1) in a Calu-3 airway epithelial cell line. 
      The goal of this study was to determine the site of actin binding on the 
      123-amino acid deltaC2-like domain. Truncations of the deltaC2-like domain were 
      made by restriction digestion and confirmed by nucleotide sequencing. His6-tagged 
      peptides were expressed in bacteria, purified, and analyzed with a Coomassie blue 
      stain for predicted size and either a 6xHis protein tag stain or an INDIA His6 
      probe for expression of the His6 tag. Truncated peptides were tested for 
      competitive inhibition of binding of activated, recombinant PKC-delta with 
      nonmuscle F-actin. Peptides from the NH2-terminal region, but not the 
      COOH-terminal region, of the deltaC2-like domain blocked binding of activated 
      PKC-delta to F-actin. The deltaC2-like domain and three NH2-terminal truncated 
      peptides of 17, 83, or 108 amino acids blocked binding, with IC50 values ranging 
      from 1.2 to 2.2 nmol (6-11 microM). NH2-terminal deltaC2-like peptides also 
      prevented methoxamine-stimulated NKCC1 activation and pulled down endogenous 
      actin from Calu-3 cells. The proximal NH2 terminus of the deltaC2-like domain 
      encodes a beta1-sheet region. The amino acid sequence of the actin-binding domain 
      is distinct from actin-binding domains in other PKC isotypes and actin-binding 
      proteins. Our results indicate that F-actin likely binds to the beta1-sheet 
      region of the deltaC2-like domain in airway epithelial cells.
FAU - Smallwood, Nicole D
AU  - Smallwood ND
AD  - Willard A. Bernbaum Cystic Fibrosis Research Center, Department of Pediatrics, 
      Rainbow Babies and Children's Hospital, and Department of Physiology and 
      Biophysics, Case Western Reserve University, Cleveland, OH 44106-4948, USA.
FAU - Hausman, Bryan S
AU  - Hausman BS
FAU - Wang, Xiangyun
AU  - Wang X
FAU - Liedtke, Carole M
AU  - Liedtke CM
LA  - eng
PT  - Journal Article
DEP - 20041208
PL  - United States
TA  - Am J Physiol Cell Physiol
JT  - American journal of physiology. Cell physiology
JID - 100901225
RN  - 0 (Actins)
RN  - 0 (Peptides)
RN  - 0 (SLC12A2 protein, human)
RN  - 0 (Sodium-Potassium-Chloride Symporters)
RN  - 0 (Solute Carrier Family 12, Member 2)
RN  - EC 2.7.11.13 (Protein Kinase C)
SB  - IM
MH  - Actins/*metabolism
MH  - Amino Acid Sequence
MH  - Animals
MH  - Binding Sites/physiology
MH  - Binding, Competitive/physiology
MH  - Cell Line
MH  - Enzyme Activation/physiology
MH  - Humans
MH  - Molecular Sequence Data
MH  - Peptides/metabolism
MH  - Protein Binding/physiology
MH  - Protein Kinase C/*chemistry/*metabolism
MH  - Respiratory Mucosa/*metabolism
MH  - Sodium-Potassium-Chloride Symporters/*metabolism
MH  - Solute Carrier Family 12, Member 2
EDAT- 2004/12/14 09:00
MHDA- 2005/04/20 09:00
CRDT- 2004/12/14 09:00
PHST- 2004/12/14 09:00 [pubmed]
PHST- 2005/04/20 09:00 [medline]
PHST- 2004/12/14 09:00 [entrez]
AID - 00484.2004 [pii]
AID - 10.1152/ajpcell.00484.2004 [doi]
PST - ppublish
SO  - Am J Physiol Cell Physiol. 2005 Apr;288(4):C906-12. doi: 
      10.1152/ajpcell.00484.2004. Epub 2004 Dec 8.