Involvement of exon 6-mediated calpastatin intracellular movements in the modulation of calpain activation.
BACKGROUND
To establish the physiological role of calpain, it is necessary to define how the protease can escape from the effect of its natural inhibitor calpastatin, since both proteins co-localize into the cell cytosol.


METHODS
To answer this question, we have overexpressed four fluorescent calpastatin constructs, differing in the composition of their XL- and L-domains, and the intracellular trafficking of this protein inhibitor has been followed by single cell fluorescence imaging.


CONCLUSIONS
By the use of these calpastatin forms differing in the type of exon-derived sequences contained in the XL- and L-domains, we have demonstrated that the sequence coded by exon 6, containing multiple phosphorylation sites, is directly involved in determining the cell localization of calpastatin. In fact, exposure to cAMP promotes the recruitment into aggregates of those calpastatin forms containing the exon 6 sequence. These protein movements are directly related to the level of cytosolic inhibitory capacity and thereby to the extent of intracellular calpain activation.


CONCLUSIONS
The recruitment of calpastatin into aggregates allows the translocation and activation of the protease to the membranes; on the contrary, the presence of large amounts of calpastatin in the cytosol prevents both processes, protecting the cell from undesired proteolysis.
De Tullio
Roberta
R
Department of Experimental Medicine (DIMES), Section of Biochemistry and Center of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 1-16132 Genova, Italy.
Cantoni
Claudia
C
Broggio
Chiara
C
Prato
Carola
C
Stifanese
Roberto
R
Averna
Monica
M
Antolini
Renzo
R
Pontremoli
Sandro
S
Melloni
Edon
E
D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000502:physiology*; D002154:Calpain / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005091:Exons; D006801:Humans; D008856:Microscopy, Fluorescence; D013347:Subcellular Fractions / Q000201:enzymology
D002135:Calcium-Binding Proteins; D017931:DNA Primers; C033668:calpastatin; D000242:Cyclic AMP; D002154:Calpain