Hsp72 interacts with paxillin and facilitates the reassembly of focal adhesions during recovery from ATP depletion.
The cytoprotective effect of heat stress proteins on epithelial cell detachment, an important cause of acute, ischemic renal failure, was examined after ATP depletion by evaluating focal adhesion complex (FAC) integrity. The intracellular distribution of FAC proteins (paxillin, talin, and vinculin) was assessed by immunohistochemistry before, during, and after exposure of renal epithelial cells to metabolic inhibitors. The resulting ATP depletion caused reversible re-distribution of all three proteins from focal adhesions to the cytosol. Paxillin, a key adaptor protein, was selected as a surrogate marker for FAC integrity in subsequent studies. Prior heat stress increased hsp72, a molecular chaperone, in both the Triton X-100-soluble and -insoluble protein fractions. Compared with ATP depleted control, heat stress significantly decreased paxillin and hsp72 shift from the Triton X-100 soluble to the insoluble protein fraction (an established marker of denaturation and aggregation); increased paxillin-hsp72 interaction detected by co-immunoprecipitation; enhanced paxillin extractability from Triton X-100-insoluble precipitates, increased the reformation of focal adhesions, and improved cell attachment (p < 0.05). To determine whether hsp72 mediates protection afforded by heat stress, cells were infected with adenovirus containing human hsp72 or empty vector. Hsp72 overexpression increased its interaction with paxillin and improved focal adhesion reformation during recovery, mimicking the protective effects of heat stress. These data suggest that hsp72 facilitates the reassembly of focal adhesions and improves cell attachment by reducing paxillin denaturation and increasing its re-solubilization after ATP depletion.
Mao
Haiping
H
Department of Nephrology, First Affiliated Hospital, Zhongshan University, GuangZhou, China 510080.
Wang
Yihan
Y
Li
Zhijian
Z
Ruchalski
Kathleen L
KL
Yu
Xueqing
X
Schwartz
John H
JH
Borkan
Steven C
SC
D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D022001:Focal Adhesions; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism; D008856:Microscopy, Fluorescence; D017830:Octoxynol / Q000494:pharmacology; D009893:Opossums; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D013347:Subcellular Fractions; D013997:Time Factors; D016596:Vinculin / Q000378:metabolism
D003598:Cytoskeletal Proteins; D003902:Detergents; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D016596:Vinculin; D000255:Adenosine Triphosphate; D017830:Octoxynol