Interaction of the recombinant S100A1 protein with twitchin kinase, and comparison with other Ca2+-binding proteins.
The giant myosin-associated twitchin kinase, a member of the Ca2+-regulated protein kinase superfamily, is activated by the EF-hand protein S100A1 in a Ca2+-dependent and Zn2+-enhanced manner. We used recombinant S100A1 to further characterize the interaction between the two proteins. Zn2+ enhanced the binding of Ca2+/S100A1 to twitchin kinase fragments (Kd < 50 nM) in assays using a BIAcore biosensor by reducing the S100A1 off rate. Other Ca2+-binding proteins (S100A6, calmodulin, and the calmodulin-like domain of Ca2+-dependent protein kinase alpha) bound to the kinase but did not activate it. These results indicate that binding of Ca2+-binding proteins alone is insufficient to trigger the intramolecular rearrangement of kinase autoinhibitory contacts required for twitchin kinase activation that is specifically elicited by the S100A1 protein. Kinase fragments that contained only the autoinhibited catalytic sequence or an additional immunoglobulin-like domain had very similar properties, indicating that the tethered immunoglobulin-like domain does not modulate kinase regulation.
Heierhorst
J
J
St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia. j.heierhorst@medicine.unimelb.edu.au
Mann
R J
RJ
Kemp
B E
BE
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015374:Biosensing Techniques; D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004800:Enzymes, Immobilized; D066298:In Vitro Techniques; D007700:Kinetics; D009124:Muscle Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D009418:S100 Proteins / Q000235:genetics / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins; D002148:Calmodulin-Binding Proteins; D017931:DNA Primers; D004800:Enzymes, Immobilized; D009124:Muscle Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D009418:S100 Proteins; C489233:unc-22 protein, C elegans; D011494:Protein Kinases