Serine/threonine protein kinases and calcium-dependent protease in senescent IMR-90 fibroblasts.
Three enzymes relevant to signal transduction were compared in replicating, quiescent and senescent human diploid fibroblasts (HDF). These were Ca(2+)-dependent thiol protease (calpain), cAMP-dependent protein kinase (Pk-A), and calcium/phospholipid-dependent protein kinase C (Pk-C). The amounts of these enzymes in quiescent HDF were slightly greater or the same as in replicating HDF. In contrast, senescent HDF exhibited higher Pk-C, Pk-A and proteolytic activities than did either replicating or quiescent cells. While the elevated protein kinase activities could be accounted for by the larger size of senescent cells relative to younger cells, the increased calpain activity exceeded this size differential. Immunoblotting studies with antisera to both Pk-C and calpain demonstrated increased enzyme concentrations in parallel with the increased activities. Photolabeling cell extracts with an analog of cAMP, 8-N3-[32P]cAMP, provides an estimate of Pk-A concentration. By this criterion, senescent HDF have more Pk-A molecules than do young cells that are either replicating or quiescent. Only the type I isozyme of Pk-A (Pk-A-I) was observed in any of these cells. Photolabeling with 8-N3-[32P]cAMP demonstrated more degradative fragments of the Pk-A regulatory subunit (RI) in senescent cells also. This is a logical consequence of the increased calpain activity in senescent cells, since RI is a substrate for calpain. These results imply that senescent cells do not fail to enter S phase owing to inadequate concentrations of Pk-A or Pk-C. Rather, the increased quantities of these enzymes in senescent cells may reflect aberrations elsewhere along signal transduction pathways that coordinate cell size with cell proliferation.
Blumenthal
E J
EJ
Molecular Toxicology Program, University of Colorado, Denver 80262.
Miller
A C
AC
Stein
G H
GH
Malkinson
A M
AM
D002118:Calcium / Q000502:physiology*; D002154:Calpain / Q000378:metabolism; D002460:Cell Line; D016922:Cellular Senescence; D004171:Diploidy; D010450:Endopeptidases / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D009994:Osmolar Concentration; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*
D013912:Threonine; D012694:Serine; D011494:Protein Kinases; D011493:Protein Kinase C; D010450:Endopeptidases; D002154:Calpain; D002118:Calcium