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Role of phosphorylation on catalytic properties of m‐calpain
Author(s) -
Wendt A S,
Thompson V F,
Mares S W,
Goll D E
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb52-d
Subject(s) - calpain , calpastatin , phosphorylation , mole , kinase , protein kinase c , biochemistry , mapk/erk pathway , chemistry , p38 mitogen activated protein kinases , microbiology and biotechnology , enzyme , biology
The intracellular protease, m‐calpain, is found in all vertebrate cells. The Ca 2+ concentrations of 300–800 μM required for half‐maximal activity of m‐calpain in in vitro assays are much higher than the 50–300 nM free [Ca 2+ ] in living cells. Hence, cells contain a mechanism to reduce the [Ca 2+ ] required for m‐calpain activity. Phosphorylation is a widely used mechanism to regulate enzyme activity. We have learned that m‐calpain in cells contains 1–3 phosphates/molecule distributed over 8 sites, 2 Tyr, 3 Ser, and 3 Thr. Several sites are in consensus protein kinase C (PKC) sequences. Moreover, evidence from two groups has suggested that m‐calpain is phosphorylated by ERK kinase and that such phosphorylation affects the catalytic properties of m‐calpain, possibly by lowering the [Ca 2+ ] required for its proteolytic activity. We have used m‐calpain purified from porcine kidney and purified ERK or PKC to learn whether these kinases would phosphorylate m‐calpain and if so, the effects of this phosphorylation on m‐calpain's catalytic properties, especially on its [Ca 2+ ] requirements. Incubation with ERK or PKC increased the phosphate content of porcine m‐calpain from 2.1 mole/mole to 6.78 mole/mole (ERK) or from 3.0 mole/mole to 3.97 mole/mole (PKC). The phosphorylated calpains had the same specific activity and Ca 2+ requirement as the control calpains that had not been incubated with the kinases. We are testing the effects of phosphorylation on inhibition of m‐calpain by calpastatin and will determine whether incubation with both kinases simultaneously or sequentially will alter the catalytic properties of this calpain. Supported NRI 2002‐35206‐11630, 2004–04338 and the MDA.