LC8 dynein light chain (DYNLL) is a highly conserved eukaryotic hub protein with dozens of binding partners and various functions beyond being a subunit of dynein and myosin Va motor proteins. Here, we compared the kinetic and thermodynamic parameters of binding of both mammalian isoforms, DYNLL1 and DYNLL2, to two putative consensus binding motifs (KXTQTX and XG(I/V)QVD) and report only subtle differences. Peptides containing either of the above motifs bind to DYNLL2 with micromolar affinity, whereas a myosin Va peptide (lacking the conserved Gln) and the noncanonical Pak1 peptide bind with K d values of 9 and 40 M, respectively. Binding of the KXTQTX motif is enthalpy-driven, although that of all other peptides is both enthalpy-and entropy-driven. Moreover, the KXTQTX motif shows strikingly slower off-rate constant than the other motifs. As most DYNLL partners are homodimeric, we also assessed the binding of bivalent ligands to DYNLL2. Compared with monovalent ligands, a significant avidity effect was found as follows: K d values of 37 and 3.5 nM for a dimeric myosin Va fragment and a Leu zipper dimerized KXTQTX motif, respectively. Ligand binding kinetics of DYNLL can best be described by a conformational selection model consisting of a slow isomerization and a rapid binding step. We also studied the binding of the phosphomimetic S88E mutant of DYNLL2 to the dimeric myosin Va fragment, and we found a significantly lower apparent K d value (3 M). We conclude that the thermodynamic and kinetic fine-tuning of binding of various ligands to DYNLL could have physiological relevance in its interaction network.

Protein phosphatase-1 (PP1) and protein phosphatase-2A (PP2A) are responsible for the dephosphorylation of the majority of phosphoserine ⁄ threonine residues in cells. In this study, we show that (-)-epigallocatechin-3-gallate (EGCG) and 1,2,3,4,6-penta-O-galloyl-b-D-glucose (PGG), polyphenolic constituents of green tea and tannins, inhibit the activity of the PP1 recombinant d-isoform of the PP1 catalytic subunit and the native PP1 catalytic subunit (PP1c) with IC 50 values of 0.47-1.35 lM and 0.26-0.4 lM, respectively. EGCG and PGG inhibit PP2Ac less potently, with IC 50 values of 15 and 6.6 lM, respectively. The structure-inhibitory potency relationships of catechin derivatives suggests that the galloyl group may play a major role in phosphatase inhibition. The interaction of EGCG and PGG with PP1c was characterized by NMR and surface plasmon resonance-based binding techniques. Competitive binding assays and molecular modeling suggest that EGCG docks at the hydrophobic groove close to the catalytic center of PP1c, partially overlapping with the binding surface of microcystin-LR or okadaic acid. This hydrophobic interaction is further stabilized by hydrogen bonding via hydroxyl ⁄ oxo groups of EGCG to PP1c residues. Comparative docking shows that EGCG binds to PP2Ac in a similar manner, but in a distinct pose. Long-term treatment (24 h) with these compounds and other catechins suppresses the viability of HeLa cells with a relative effectiveness reminiscent of their in vitro PP1c-inhibitory potencies. The above data imply that the phosphatase-inhibitory features of these polyphenols may be implicated in the wide spectrum of their physiological influence.Abbreviations CLA, calyculin-A; EC, (-)-epicatechin; ECG, (-)-epicatechin-3-gallate; EGC, (-)-epigallocatechin; EGCG, (-)-epigallocatechin-3-gallate; ERK, extracellular signal-related kinase; GCG, (-)-gallocatechin-3-gallate; JNK, c-Jun NH 2 -terminal kinase; MC-LR, microcystin-LR; MLC20, 20-kDa myosin light chain; MP, myosin phosphatase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; MYPT1, myosin phosphatase target subunit-1; OA, okadaic acid; PGG, 1,2,3,4,6-penta-O-galloyl-b-D-glucose; PP1, protein phosphatase-1; PP1c, protein phosphatase-1 catalytic subunit; PP1cd, hexahistidine-tagged recombinant protein phosphatase-1 catalytic subunit d-isoform; PP2A, protein phosphatase-2A; PP2Ac, protein phosphatase-2A catalytic subunit; RU, response unit; SPR, surface plasmon resonance; STD, saturation transfer difference; TM, tautomycin.

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TIMAP, TGF-β inhibited, membrane-associated protein, is highly abundant in endothelial cells (EC). We have shown earlier the involvement of TIMAP in PKA-mediated ERM (ezrin-radixin-moesin) dephosphorylation as part of EC barrier protection by TIMAP (Csortos et al., 2008). Emerging data demonstrate the regulatory role of TIMAP on protein phosphatase 1 (PP1) activity. We provide here evidence for specific interaction (Ka=1.80×106 M−1) between non-phosphorylated TIMAP and the catalytic subunit of PP1 (PP1c) by surface plasmon resonance based binding studies. Thiophosphorylation of TIMAP by PKA, or sequential thiophosphorylation by PKA and GSK3β slightly modifies the association constant for the interaction of TIMAP with PP1c and decreases the rate of dissociation. However, dephosphorylation of phospho-moesin substrate by PP1cβ is inhibited to different extent in the presence of non- (~60% inhibition), mono- (~50% inhibition) or double-thiophosphorylated (<10% inhibition) form of TIMAP. Our data suggest that double-thiophosphorylation of TIMAP has minor effect on its binding ability to PP1c, but considerably attenuates its inhibitory effect on the activity of PP1c. PKA activation by forskolin treatment of EC prevented thrombin evoked barrier dysfunction and ERM phosphorylation at the cell membrane (Csortos et al., 2008). With the employment of specific GSK3β inhibitor it is shown here that PKA activation is followed by GSK3β activation in bovine pulmonary EC and both of these activations are required for the rescuing effect of forskolin in thrombin treated EC. Our results suggest that the forskolin induced PKA/ GSK3β activation protects the EC barrier via TIMAP-mediated decreasing of the ERM phosphorylation level.