SERINE THREONINE PROTEIN-KINASES AND CALCIUM-DEPENDENT PROTEASE IN SENESCENT IMR-90 FIBROBLASTS

Three enzymes relevant to signal transduction were compared in replica ting, quiescent and senescent human diploid fibroblasts (HDF). These w ere Ca2+-dependent thiol protease (calpain), cAMP-dependent protein ki nase (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 exhibit ed higher Pk-C, Pk-A and proteolytic activities than did either replic ating 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 d ifferential. Immunoblotting studies with antisera to both Pk-C and cal pain demonstrated increased enzyme concentrations in parallel with the increased activities. Photolabeling cell extracts with an analog of c AMP, 8-N3-[P-32]cAMP, provides an estimate of Pk-A concentration. By t his criterion, senescent HDF have more Pk-A molecules than do young ce lls 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 wit h 8-N3-[P-32]cAMP demonstrated more degradative fragments of the Pk-A regulatory subunit (RI) in senescent cells also. This is a logical con sequence of the increased calpain activity in senescent cells, since R I 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 senes cent cells may reflect aberrations elsewhere along signal transduction pathways that coordinate cell size with cell proliferation.