Recent molecular cloning experiments have identified a 25 amino-acid region as the calmodulin-binding domain of the alpha-subunit of rat brain Ca2+/calmodulin-dependent multifunctional protein kinase II (CaM-K II). Synthetic peptides, derived from the deduced amino-acid sequence encompassing this region, were examined for their ability to bind calmodulin in a calcium dependent manner and to inhibit the Ca2+/calmodulin-dependent autophosphorylation of CaM-K II. Comparison of these structure-function relationships highlighted a region of 5 amino-acids, which was essential for calmodulin interaction and inhibition of kinase activity. This region demonstrated some homology with other calmodulin-binding peptides, and may represent a key site of interaction of the kinase with calmodulin. These analyses provide additional insight into the molecular mechanism underlying the Ca2+ regulation of CaM-K II.
"Most protein kinases are capable of autophosphorylation , and autophosphorylation activity is frequently employed as a test for kinase activity . To determine whether ILK can undergo autophosphorylation, we carried out kinase reactions in the absence of exogenous substrates. "
[Show abstract][Hide abstract] ABSTRACT: Background
Integrin-linked kinase (ILK) is a highly evolutionarily conserved, multi-domain signaling protein that localizes to focal adhesions, myofilaments and centrosomes where it forms distinct multi-protein complexes to regulate cell adhesion, cell contraction, actin cytoskeletal organization and mitotic spindle assembly. Numerous studies have demonstrated that ILK can regulate the phosphorylation of various protein and peptide substrates in vitro, as well as the phosphorylation of potential substrates and various signaling pathways in cultured cell systems. Nevertheless, the ability of ILK to function as a protein kinase has been questioned because of its atypical kinase domain.Methodology/Principal FindingsHere, we have expressed full-length recombinant ILK, purified it to >94% homogeneity, and characterized its kinase activity. Recombinant ILK readily phosphorylates glycogen synthase kinase-3 (GSK-3) peptide and the 20-kDa regulatory light chains of myosin (LC20). Phosphorylation kinetics are similar to those of other active kinases, and mutation of the ATP-binding lysine (K220 within subdomain 2) causes marked reduction in enzymatic activity. We show that ILK is a Mn-dependent kinase (the Km for MnATP is ∼150-fold less than that for MgATP).Conclusions/SignificanceTaken together, our data demonstrate that ILK is a bona fide protein kinase with enzyme kinetic properties similar to other active protein kinases.
PLoS ONE 08/2010; 5(8). DOI:10.1371/journal.pone.0012356 · 3.23 Impact Factor
"The fact that Mg 2+ , if anything, impairs ATP binding by the CASK CaM-kinase domain is consistent with the notion that this domain is indeed a `pseudokinase'; despite its constitutively active conformation and avid ATP binding. To test this, we examined whether the CASK CaMkinase domain exhibits autophosphorylation activity, which is typical for CaM-kinases and can be employed as a test for kinase activity (Hanley et al., 1988). "
[Show abstract][Hide abstract] ABSTRACT: CASK is a unique MAGUK protein that contains an N-terminal CaM-kinase domain besides the typical MAGUK domains. The CASK CaM-kinase domain is presumed to be a catalytically inactive pseudokinase because it lacks the canonical DFG motif required for Mg2+ binding that is thought to be indispensable for kinase activity. Here we show, however, that CASK functions as an active protein kinase even without Mg2+ binding. High-resolution crystal structures reveal that the CASK CaM-kinase domain adopts a constitutively active conformation that binds ATP and catalyzes phosphotransfer without Mg2+. The CASK CaM-kinase domain phosphorylates itself and at least one physiological interactor, the synaptic protein neurexin-1, to which CASK is recruited via its PDZ domain. Thus, our data indicate that CASK combines the scaffolding activity of MAGUKs with an unusual kinase activity that phosphorylates substrates recuited by the scaffolding activity. Moreover, our study suggests that other pseudokinases (10% of the kinome) could also be catalytically active.
"Because CaM is an important and ubiquitous target of free calcium, i.e., Ca 2ϩ /CaM formation during Ca 2ϩ increases, the role of Ca 2ϩ /CaM signal pathways in FK-506-induced synaptic potentiation was examined. We first used a high-affinity CBP (Hanley et al., 1988; Kelly et al., 1989) to block intracellular cascades initially triggered by Ca 2ϩ /CaM. Microelectrodes containing 100 M CBP plus 50 M FK-506 in 2 M KAc were used for intracellular recordings and postsynaptic co-injections into CA1 neurons. "
[Show abstract][Hide abstract] ABSTRACT: Protein phosphorylation and dephosphorylation are believed to functionally couple neuronal activity and synaptic plasticity. Our previous results indicated that postsynaptic Ca2+/calmodulin (CaM) signaling pathways play an important role in setting synaptic strength, and calcineurin (CaN) activity limits synaptic responses during basal synaptic transmission and long-term potentiation expression. The inhibition of postsynaptic CaN activity by FK-506 or an autoinhibitory peptide induced synaptic potentiation in hippocampal slices, which occludes tetanus-induced LTP. FK-506-induced synaptic potentiation was expressed in adult but not young rats. To elucidate mechanisms underlying CaN-inhibited synaptic potentiation, we co-injected certain agents affecting Ca2+ signaling pathways with CaN inhibitors into CA1 neurons. Synaptic potentiation induced by FK-506 was significantly attenuated by co-injecting BAPTA, heparin/dantrolene (inhibitors of intracellular Ca2+ release), a CaM-binding peptide, or CaM-KII/PKC pseudosubstrate peptides. These results indicate that postsynaptic CaN activity can downregulate evoked synaptic transmission by weakening intracellular Ca2+ signals and downstream protein kinase activities.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 07/1997; 17(12):4600-11. · 6.34 Impact Factor
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