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Age-dependent changes in murine protein kinase and protease enzymes
E J Blumenthal1, A M Malkinson
1Molecular and Environmental Toxicology Program, School of Pharmacy, University of Colorado, Boulder 80309-0297.
Abstract:
Hormone responsiveness is mediated by signal transduction mechanisms involving second messengers, such as cAMP and Ca2+, which regulate reversible changes in the phosphorylation state of proteins. During senescence individuals frequently exhibit a diminished responsiveness to hormones. We examined changes in enzymes involved in protein phosphorylation reactions that might account for this decreased adaptiveness in old mice, and observed the following post-maturational changes: (1) cAMP-dependent protein kinase (Pk-A) specific activity decreased in spleen cytosol and in the particulate fractions of lung, spleen and liver of 24-month-old mice as compared to 2-month-old mice. Splenic cytosolic Pk-A activity decreased by 18 months of age, while particulate activity decreased by 6 months; (2) The amount of 8-N3-[32P]cAMP, a photoaffinity analog of cAMP, incorporated into Pk-A regulatory (R)-subunits from spleen and liver particulate fractions decreased, while photolabeling of R-subunit degradative products with this analog in heart and spleen cytosol increased. (3) Age-dependent increases in membrane-associated protease activities were found in all organs, along with a decrease in cytosolic lung calpain activity. These proteolytic changes may account for the enhanced R-subunit degradation and decreased Pk-A activities observed during senescence. (4) Age-dependent alterations in Ca2+/phospholipid-dependent protein kinase (Pk-C) are organ specific: lung, liver, brain, and heart demonstrate no change in Pk-C activity, while spleen exhibits decreased activity. We hypothesize that these age-dependent alterations in kinase and proteolytic activities may be in part responsible for changes in cellular response to hormonal stimulation, differentiation signals, and antigen responsiveness during senescence.
Insights
Aging reduces hormone responsiveness by altering protein phosphorylation enzymes like cAMP-dependent protein kinase (Pk-A). Proteolytic activity increases, degrading Pk-A subunits, contributing to diminished cellular signaling in aged mice.
Area of Science:
- Biochemistry
- Cellular Biology
- Gerontology
Background:
- Hormone responsiveness relies on signal transduction pathways involving second messengers like cAMP and Ca2+.
- Cellular signaling and responsiveness to hormones often diminish with age (senescence).
Purpose of the Study:
- To investigate age-related changes in protein phosphorylation enzymes that may explain reduced hormone responsiveness in aging mice.
- To identify specific alterations in kinases and proteases contributing to senescence-associated signaling deficits.
Main Methods:
- Compared enzyme activities (cAMP-dependent protein kinase (Pk-A), Ca2+/phospholipid-dependent protein kinase (Pk-C)) and protein degradation in young (2-month-old) versus old (24-month-old) mice.
- Utilized photoaffinity labeling with a cAMP analog to assess Pk-A regulatory subunit binding and degradation.
- Measured membrane-associated and cytosolic protease activities across various organs.
Main Results:
- Decreased Pk-A specific activity was observed in multiple organs of aged mice.
- Increased degradation of Pk-A regulatory subunits was evident in aged mice, linked to elevated protease activity.
- Ca2+/phospholipid-dependent protein kinase (Pk-C) activity showed organ-specific changes, decreasing in the spleen.
Conclusions:
- Age-dependent alterations in protein kinase and protease activities likely contribute to reduced cellular responsiveness to hormonal and other signaling stimuli during senescence.
- These molecular changes in signaling pathways may underlie functional declines observed in aging individuals.