Expression of the major protein kinase C substrate, the acidic 80-kilodalton myristoylated alanine-rich C kinase substrate, increases sharply when Swiss 3T3 cells move out of cycle and enter G0.
The expression of the major protein kinase C (PKC) substrate, originally called "80K" for acidic SDS/PAGE-observed 80-kDa PKC substrate and now called "MARCKS" for myristoylated alanine-rich C kinase substrate, in Swiss 3T3 fibroblasts changes strikingly (15- to 22-fold) during transitions of cell growth. Quiescent cells in G0 express high levels of MARCKS mRNA and protein. However, plating these cells in fresh medium at low density to stimulate multiple rounds of cell division caused a striking down-regulation of MARCKS expression. The mRNA level declined to a minimum of 4.5% compared with quiescent control cells 6 hr after plating, and protein levels declined during the same period to 6.5% of the control value. This rapid down-regulation was independent of PKC activation and length of exposure to trypsin (1-10 min) but required plating in medium containing fresh serum. MARCKS mRNA and protein levels remained down-regulated for 3 days, during which time the cells were actively progressing through the cell cycle as judged by fluorescence-activated cell sorting analysis. However, on reaching quiescence, the expression of MARCKS mRNA and protein increased markedly. Furthermore, the rate of recovery of MARCKS mRNA and protein levels was shown to be dependent on the supply of serum-derived growth factors in the medium. Addition of hydroxyurea to arrest the cells in S phase or at the G1/S boundary rather than G0 completely prevented the recovery of MARCKS protein. The down-regulation of MARCKS following plating and its serum-dependent recovery was also demonstrated in tertiary cultures of mouse embryo fibroblasts. The results suggest that MARCKS may play a role in the regulation of entry and exit of cells from G0.
Herget
T
T
Imperial Cancer Research Fund, London, United Kingdom.
Brooks
S F
SF
Broad
S
S
Rozengurt
E
E
D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002453:Cell Cycle / Q000502:physiology*; D002455:Cell Division / Q000502:physiology; D005434:Flow Cytometry; D016193:G1 Phase / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects; D006133:Growth Substances / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008565:Membrane Proteins; D051379:Mice; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016192:Resting Phase, Cell Cycle / Q000502:physiology; D015183:Restriction Mapping; D016196:S Phase / Q000502:physiology; D013997:Time Factors
D006133:Growth Substances; D047908:Intracellular Signaling Peptides and Proteins; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D011506:Proteins; D012333:RNA, Messenger; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C