CaMKII‐dependent filamin phosphorylation attenuates the MARCKS regulation of ENaC (892.24)

• Published in: The FASEB journal Vol. 28; no. S1
• Main Authors: Alli, Abdel, Bao, Hui‐Fang, Montgomery, Darrice, Ghant, Marcus, Al‐Khalili, Otor, Eaton, Douglas
• Format: Journal Article
• Language: English
• Published: The Federation of American Societies for Experimental Biology 01-04-2014
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.28.1_supplement.892.24

Filamin A is an actin crosslinking protein regulated by phosphorylation. Several kinases including protein kinase A (PKA), protein kinase B (Akt), protein kinase C (PKC), and Ca(2+)/calmodulin‐dependent protein kinase II (CaMKII) phosphorylate filamin A. Xenopus laevis distal nephron epithelial (2F3) cells pretreated with the specific CaMKII inhibitor, KN‐93, showed an increase in amiloride‐sensitive transepithelial current calculated from transepithelial voltage and resistance measurements obtained with a volt‐ohmmeter, and an increase in ENaC open probability measured by single‐channel patch clamp studies. Whole cell lysates from 2F3 cells pretreated with KN‐93 or vehicle control were used to immunoprecipitate filamin, phosphofilamin, MARCKS, and ENaC before immunoblotting for each protein using specific antibodies. Immunoblot and densitometric analysis showed the level of filamin phosphorylation decreased with KN‐93 treatment compared to vehicle control. The association between ENaC and MARCKS was augmented in cells treated with KN‐93 compared to cells treated with vehicle only. ENaC‐MARCKS interaction is necessary for normal ENaC activity. Sucrose density gradient fractions from KN‐93 or vehicle treated cells were analyzed by immunoblotting. Similar to various actin cytoskeleton proteins, filamin protein expression was observed in non‐lipid raft cytoplasmic fractions. The expression of phosphofilamin between these fractions was different between the KN‐93 treated group compared to the vehicle treated control group. Taken together, these studies indicate filamin is an integral component of the actin cytoskeleton and phosphorylation of filamin by CaMKII disrupts the interaction between MARCKS and ENaC.
Grant Funding Source: DK093255‐01