Interaction of the Recombinant S100A1 Protein with Twitchin Kinase, and Comparison with Other Ca²⁺‐Binding Proteins

Abstract

The giant myosin-associated twitchin kinase, a member of the Ca'+-regulated protein kinase superfamily, is activated by the EF-hand protein SlOOAl in a Ca2+-dependent and Zn'+-enhanced manner. We used recombinant SlOOAl to further characterize the interaction between the two proteins. ZnZ' enhanced the binding of CaZ+/SIOOA1 to twitchin kinase fragments (K, < SO nM) in assays using a BIAcore biosensor by reducing the SlOOAl off rate. Other Ca2+-binding proteins (SlOOA6, calmodulin, and the calmodulin-like domain of Ca2+-dependent protein kinase a) 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 SlOOAl 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.Keywords: protein kinase; twitchin ; titin; ,5100; calmodulin.Intracellular second messenger functions of calcium ions are mediated by specific calcium-binding proteins [I]. Members of a large protein superfamily bind calcium via the helix-loop-helix EF-hand motif (21. Calmodulin (CaM), the most thoroughly characterized EF-hand protein, associates with its effector enzymes (such as CaM-dependent protein kinases, the phosphatase calcineurin or phosphodiesterase) and regulates their activity upon calcium binding [3, 41. Members of the subfamily of S100-like EF-hand proteins are encoded by at least 16 different genes, most of them clustered on human chromosome lq21 [S, 61. The SlOO family differs from CaM in two important aspects. First, in contrast to CaM that is monomeric and contains four EF-hands/molecule, S100 proteins have only two EF-hands/= 100 amino-acid-residue subunit, but are dimeric; second, while CaM is ubiquitously expressed, S 100 proteins are generally expressed in a tissue-specific and often developmentally regulated manner [5, 61. Some SlOO proteins are reported to have extracellular functions as neurotrophic (e.g. SlOOS) or chemotactic factors (e.g. SIOOAS, SlOOA9), whereas others act intracellularly either as Ca' ' buffer/transport proteins ( e g calbindin) or, similarly to CaM, as proposed trigger proteins that associate with effector proteins to transduce intracellular Ca'+ signals [5, 61.The current understanding of the effector functions of SlOO proteins is limited by the fact that only few targets have been identified. Furthermore, even fewer of the targets are effector enzymes whose activity is significantly regulated by Ca2+/S100 proteins [S-7]. So far, the enzyme most dramatically affected by an S100-like protein is the myosin-associated giant protein kinase twitchin [S], a member of the myosin light-chain kinase family. CaZ+/SlOOA1, but not Ca2+/CaM, stimulates the twitchin kinase V,,, by more than 1000-fold [...

Divalent Metal Ion Complexes of S100B in the Absence and Presence of Pentamidine

“…14 Likewise, target protein interactions involving S100A6 and S100A11 were of higher affinity when these S100 proteins were bound to Zn 2+ . 12 For S100A1, Zn 2+ binding provided more than a 30-fold enhancement in binding the protein target twitchen kinase 52 and raised the question of whether Zn 2+ binding modulates S100 protein function as part of a more general mechanism in vivo for this family of proteins. 4,47 Close examination of conformational change that Zn 2+ induced in Ca 2+ -S100B (Fig.…”

Impaired Cardiac Contractility Response to Hemodynamic Stress in S100A1-Deficient Mice

“…The S100A1 gene was isolated from a mouse 129Sv genomic DNA library (3) in the -DASH II cloning vector by hybridization of plaque lifts using a radiolabeled rat S100A1 cDNA probe (18). Restriction fragments were subcloned into pBluescript vectors for mapping and sequence analysis.…”

The Giant Protein AHNAK Is a Specific Target for the Calcium- and Zinc-binding S100B Protein

“…The interaction of small EF-hand calcium binding proteins with giant proteins has a precedent in the literature. Two giant protein kinases, titin (3000 kDa) and twitchin (750 kDa), interact with Ca 2ϩ -CaM and Ca 2ϩ /Zn 2ϩ -S100A1, respectively (36). However, no kinase domain is predicted based on AHNAK structure.…”