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Synthesis, degradation, and subcellular localization of proteins encoded by the primary response genes TIS7/PC4 and
B C Varnum1, S T Reddy, R A Koski
1Amgen, Inc., Thousand Oaks, California 91320.
Abstract:
Murine TIS7 and TIS21 cDNAs were cloned from phorbol ester-induced Swiss 3T3 cells. The cognate rat cDNAs, PC4 and PC3, were cloned from nerve growth factor (NGF)-treated PC12 pheochromocytoma cells. The TIS7/PC4 and TIS21/PC3 primary response genes are rapidly and transiently induced in response to serum, phorbol esters, and polypeptide growth factors in quiescent Swiss 3T3 cells and by NGF and other ligands in PC12 cells. In both 3T3 and PC12 cells the appearance of the TIS21/PC3 message precedes that of TIS7/PC4 message following ligand stimulation, suggesting that the TIS21/PC3 protein is likely to be synthesized more rapidly than the TIS7/PC4 protein. Using antisera prepared against recombinant TIS21 and TIS7 proteins, we find that the TIS21/PC3 protein is, indeed, synthesized more rapidly than the TIS7/PC4 protein following stimulation in both 3T3 and PC12 cells. In addition, "pulse-chase" experiments demonstrate that the TIS21/PC3 protein is degraded much more rapidly than the TIS7/PC4 protein. The sequences of the predicted PC3 and PC4 proteins have lead to the speculation that these two proteins may both be secreted from cells following stimulation. The PC4 protein is reported to have some sequence similarity to interferons. The TIS21/PC3 protein contains a presumptive leader sequence. Using our antisera to the recombinant proteins, however, we cannot detect secretion of radiolabelled TIS7/PC4 or TIS21/PC3 protein. Immunohistochemical and subcellular fractionation experiments suggest that the TIS7 protein is a membrane associated, non-nuclear intracellular protein. The TIS21 protein, in contrast, is a non-nuclear, soluble intracellular protein.
Insights
Primary response genes TIS7/PC4 and TIS21/PC3 are rapidly induced by growth factors. TIS21/PC3 protein is synthesized and degraded faster than TIS7/PC4, with TIS21 being soluble and TIS7 membrane-associated intracellular proteins.
Area of Science:
- Molecular biology
- Cell signaling
- Gene expression
Background:
- Primary response genes are rapidly induced by extracellular stimuli.
- TIS7/PC4 and TIS21/PC3 are homologous primary response genes identified in murine and rat cells, respectively.
- These genes are activated by various growth factors and ligands.
Purpose of the Study:
- To investigate the synthesis, degradation, and subcellular localization of TIS7/PC4 and TIS21/PC3 proteins.
- To compare the kinetics of TIS21/PC3 and TIS7/PC4 protein expression and turnover.
- To determine if these proteins are secreted or remain intracellular.
Main Methods:
- Cloning of murine TIS7 and TIS21 cDNAs, and rat PC4 and PC3 cDNAs.
- Ligand stimulation of Swiss 3T3 and PC12 cells.
- Antisera production against recombinant TIS21 and TIS7 proteins.
- Pulse-chase experiments to assess protein degradation.
- Immunohistochemistry and subcellular fractionation to determine protein localization.
Main Results:
- TIS21/PC3 mRNA and protein expression precede TIS7/PC4 following stimulation.
- TIS21/PC3 protein is synthesized and degraded more rapidly than TIS7/PC4 protein.
- Neither TIS7/PC4 nor TIS21/PC3 proteins were detected as secreted.
- TIS7 protein is membrane-associated and intracellular; TIS21 protein is soluble and intracellular.
Conclusions:
- TIS21/PC3 and TIS7/PC4 proteins exhibit distinct synthesis and degradation kinetics.
- TIS21/PC3 and TIS7/PC4 proteins are intracellular, with TIS21 being soluble and TIS7 membrane-associated.
- These findings provide insights into the post-translational regulation and cellular fate of primary response proteins.