Regulation of calpain B from Drosophila melanogaster by phosphorylation.
Calpain B is one of the two catalytically competent calpain (calcium-activated papain) isoenzymes in Drosophila melanogaster. Because structural predictions hinted at the presence of several potential phosphorylation sites in this enzyme, we investigated the in vitro phosphorylation of the recombinant protein by protein kinase A as well as by the extracellular signal-regulated protein kinases (ERK) 1 and 2. By MS, we identified Ser845 in the Ca2+ binding region of an EF-hand motif, and Ser240 close to the autocatalytic activation site of calpain B, as being the residues phosphorylated by protein kinase A. In the transducer region of the protease, Thr747 was shown to be the target of the ERK phosphorylation. Based on the results of three different assays, we concluded that the treatment of calpain B with protein kinase A and ERK1 and ERK2 kinases increases the rate of the autoproteolytic activation of the enzyme, together with the rate of the digestion of external peptide or protein substrates. Phosphorylation also elevates the Ca2+ sensitivity of the protease. The kinetic analysis of phosphorylation mimicking Thr747Glu and Ser845Glu calpain B mutants confirmed the above conclusions. Out of the three phosphorylation events tested in vitro, we verified the in vivo phosphorylation of Thr747 in epidermal growth factor-stimulated Drosophila S2 cells. The data obtained suggest that the activation of the ERK pathway by extracellular signals results in the phosphorylation and activation of calpain B in fruit flies.
Kovács
László
L
Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Hungary.
Alexa
Anita
A
Klement
Eva
E
Kókai
Endre
E
Tantos
Agnes
A
Gógl
Gergö
G
Sperka
Tamás
T
Medzihradszky
Katalin F
KF
Tözsér
József
J
Dombrádi
Viktor
V
Friedrich
Péter
P
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D029721:Drosophila Proteins; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; C485092:CalpB protein, Drosophila; D002154:Calpain; D002118:Calcium