Cleavage of the actin-capping protein alpha -adducin at Asp-Asp-Ser-Asp633-Ala by caspase-3 is preceded by its phosphorylation on serine 726 in cisplatin-induced apoptosis of renal epithelial cells.
Decreased phosphorylation of focal adhesion kinase and paxillin is associated with loss of focal adhesions and stress fibers and precedes the onset of apoptosis (van de Water, B., Nagelkerke, J. F., and Stevens, J. L. (1999) J. Biol. Chem. 274, 13328-13337). The cortical actin cytoskeletal network is also lost during apoptosis, yet little is known about the temporal relationship between altered phosphorylation of proteins that are critical in the regulation of this network and their potential cleavage by caspases during apoptosis. Adducins are central in the cortical actin network organization. Cisplatin caused apoptosis of renal proximal tubular epithelial cells, which was associated with the cleavage of alpha-adducin into a 74-kDa fragment; this was blocked by a general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk). Hemagglutinin-tagged human alpha-adducin was cleaved into a similar 74-kDa fragment by caspase-3 in vitro but not by caspase-6 or -7. Asp-Arg-Val-Asp(29)-Glu, Asp-Ile-Val-Asp(208)-Arg, and Asp-Asp-Ser-Asp(633)-Ala were identified as the principal caspase-3 cleavage sites; Asp-Asp-Ser-Asp(633)-Ala was key in the formation of the 74-kDa fragment. Cisplatin also caused an increased phosphorylation of alpha-adducin and gamma-adducin in the MARCKS domain that preceded alpha-adducin cleavage and was associated with loss of adducins from adherens junctions; this was not affected by z-VAD-fmk. In conclusion, the data support a model in which increased phosphorylation of alpha-adducin due to cisplatin leads to dissociation from the cytoskeleton, a situation rendered irreversible by caspase-3-mediated cleavage of alpha-adducin at Asp-Asp-Ser-Asp(633)-Ala.
van de Water
B
B
Division of Toxicology, Leiden Amsterdam Center for Drug Research, Leiden University, The Netherlands. b.water@lacdr.leidenuniv.nl
Tijdens
I B
IB
Verbrugge
A
A
Huigsloot
M
M
Dihal
A A
AA
Stevens
J L
JL
Jaken
S
S
Mulder
G J
GJ
D000199:Actins / Q000378:metabolism; D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D019556:COS Cells; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death; D002945:Cisplatin / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007668:Kidney / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D013049:Spectrin; D000099291:Adducins
D000199:Actins; D000590:Amino Acid Chloromethyl Ketones; D000970:Antineoplastic Agents; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D053148:Caspase 3; D020169:Caspases; D002945:Cisplatin; D007770:L-Lactate Dehydrogenase; D008840:Microfilament Proteins; D020033:Protein Isoforms; D011493:Protein Kinase C; D012694:Serine; D013049:Spectrin; D000099291:Adducins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C505407:CASP3 protein, human; C505410:Casp3 protein, rat