Osmotic shrinkage elicits FAK- and Src phosphorylation and Src-dependent NKCC1 activation in NIH3T3 cells.
The mechanisms linking cell volume sensing to volume regulation in mammalian cells remain incompletely understood. Here, we test the hypothesis that activation of nonreceptor tyrosine kinases Src, focal adhesion kinase (FAK), and Janus kinase-2 (Jak2) occurs after osmotic shrinkage of NIH3T3 fibroblasts and contributes to volume regulation by activation of NKCC1. FAK phosphorylation at Tyr397, Tyr576/577, and Tyr861 was increased rapidly after exposure to hypertonic (575 mOsm) saline, peaking after 10 (Tyr397, Tyr576/577) and 10-30 min (Tyr861). Shrinkage-induced Src family kinase autophosphorylation (pTyr416-Src) was induced after 2-10 min, and immunoprecipitation indicated that this reflected phosphorylation of Src itself, rather than Fyn and Yes. Phosphorylated Src and FAK partly colocalized with vinculin, a focal adhesion marker, after hypertonic shrinkage. The Src inhibitor pyrazolopyrimidine-2 (PP2, 10 μM) essentially abolished shrinkage-induced FAK phosphorylation at Tyr576/577 and Tyr861, yet not at Tyr397, and inhibited shrinkage-induced NKCC1 activity by ∼50%. The FAK inhibitor PF-573,228 augmented shrinkage-induced Src phosphorylation, and inhibited shrinkage-induced NKCC1 activity by ∼15%. The apparent role of Src in NKCC1 activation did not reflect phosphorylation of myosin light chain kinase (MLC), which was unaffected by shrinkage and by PP2, but may involve Jak2, a known target of Src, which was rapidly activated by osmotic shrinkage and inhibited by PP2. Collectively, our findings suggest a major role for Src and possibly the Jak2 axis in shrinkage-activation of NKCC1 in NIH3T3 cells, whereas no evidence was found for major roles for FAK and MLC in this process.
Department of Biology, University of Copenhagen, Copenhagen, Denmark.
Line Jee Hartmann
LJ
Rasmussen
Department of Biology, University of Copenhagen, Copenhagen, Denmark.
Helene Steenkær Holm
HS
Müller
Department of Biology, University of Copenhagen, Copenhagen, Denmark.
Bente
B
Jørgensen
Department of Biology, University of Copenhagen, Copenhagen, Denmark sfpedersen@bio.ku.dk.
Stine Falsig
SF
Pedersen
Department of Biology, University of Copenhagen, Copenhagen, Denmark.
Else Kay
EK
Hoffmann
D000818:Animals; D002460:Cell Line; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D053614:Janus Kinase 2 / Q000378:metabolism; D051379:Mice; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D041681:NIH 3T3 Cells; D009995:Osmosis / Q000502:physiology*; D010766:Phosphorylation / Q000502:physiology*; D064506:Solute Carrier Family 12, Member 2 / Q000378:metabolism*; D016596:Vinculin / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
C578878:Slc12a2 protein, mouse; D064506:Solute Carrier Family 12, Member 2; D016596:Vinculin; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; C496051:Ptk2 protein, mouse; D019061:src-Family Kinases; D009219:Myosin-Light-Chain Kinase
FAK phosphorylation; Jak2; Na+-K+-2Cl− cotransport; SLC12A1; Src phosphorylation; focal adhesions; vinculin