Non-catalytic N-terminal Domain Research Articles

Molecular mechanism for regulating APOBEC3G DNA editing function by the non-catalytic domain

APOBEC3G, part of the AID/APOBEC cytidine deaminase family, is crucial for antiviral immunity. It has two zinc-coordinated cytidine-deaminase domains. The non-catalytic N-terminal domain strongly binds to nucleic acids, whereas the C-terminal domain catalyzes C-to-U editing in single-stranded DNA. The interplay between the two domains is not fully understood. Here, we show that DNA editing function of rhesus macaque APOBEC3G on linear and hairpin loop DNA is enhanced by AA or GA dinucleotide motifs present downstream in the 3’-direction of the target-C editing sites. The effective distance between AA/GA and the target-C sites is contingent on the local DNA secondary structure. We present two co-crystal structures of rhesus macaque APOBEC3G bound to ssDNA containing AA and GA, revealing the contribution of the non-catalytic domain in capturing AA/GA DNA. Our findings elucidate the molecular mechanism of APOBEC3G’s cooperative function, which is critical for its antiviral role and its contribution to mutations in cancer genomes.

Structural and mechanistic characterization of bifunctional heparan sulfate N-deacetylase-N-sulfotransferase 1

Heparan sulfate (HS) polysaccharides are major constituents of the extracellular matrix, which are involved in myriad structural and signaling processes. Mature HS polysaccharides contain complex, non-templated patterns of sulfation and epimerization, which mediate interactions with diverse protein partners. Complex HS modifications form around initial clusters of glucosamine-N-sulfate (GlcNS) on nascent polysaccharide chains, but the mechanistic basis underpinning incorporation of GlcNS itself into HS remains unclear. Here, we determine cryo-electron microscopy structures of human N-deacetylase-N-sulfotransferase (NDST)1, the bifunctional enzyme primarily responsible for initial GlcNS modification of HS. Our structures reveal the architecture of both NDST1 deacetylase and sulfotransferase catalytic domains, alongside a non-catalytic N-terminal domain. The two catalytic domains of NDST1 adopt a distinct back-to-back topology that limits direct cooperativity. Binding analyses, aided by activity-modulating nanobodies, suggest that anchoring of the substrate at the sulfotransferase domain initiates the NDST1 catalytic cycle, providing a plausible mechanism for cooperativity despite spatial domain separation. Our data shed light on key determinants of NDST1 activity, and describe tools to probe NDST1 function in vitro and in vivo.

Exportin-mediated nucleocytoplasmic transport maintains Pch2 homeostasis during meiosis.

The meiotic recombination checkpoint reinforces the order of events during meiotic prophase I, ensuring the accurate distribution of chromosomes to the gametes. The AAA+ ATPase Pch2 remodels the Hop1 axial protein enabling adequate levels of Hop1-T318 phosphorylation to support the ensuing checkpoint response. While these events are localized at chromosome axes, the checkpoint activating function of Pch2 relies on its cytoplasmic population. In contrast, forced nuclear accumulation of Pch2 leads to checkpoint inactivation. Here, we reveal the mechanism by which Pch2 travels from the cell nucleus to the cytoplasm to maintain Pch2 cellular homeostasis. Leptomycin B treatment provokes the nuclear accumulation of Pch2, indicating that its nucleocytoplasmic transport is mediated by the Crm1 exportin recognizing proteins containing Nuclear Export Signals (NESs). Consistently, leptomycin B leads to checkpoint inactivation and impaired Hop1 axial localization. Pch2 nucleocytoplasmic traffic is independent of its association with Zip1 and Orc1. We also identify a functional NES in the non-catalytic N-terminal domain of Pch2 that is required for its nucleocytoplasmic trafficking and proper checkpoint activity. In sum, we unveil another layer of control of Pch2 function during meiosis involving nuclear export via the exportin pathway that is crucial to maintain the critical balance of Pch2 distribution among different cellular compartments.

Structural and dynamic basis of DNA capture and translocation by mitochondrial Twinkle helicase.

Twinkle is a mitochondrial replicative helicase which can self-load onto and unwind mitochondrial DNA. Nearly 60 mutations on Twinkle have been linked to human mitochondrial diseases. Using cryo-electron microscopy (cryo-EM) and high-speed atomic force microscopy (HS-AFM), we obtained the atomic-resolution structure of a vertebrate Twinkle homolog with DNA and captured in real-time how Twinkle is self-loaded onto DNA. Our data highlight the important role of the non-catalytic N-terminal domain of Twinkle. The N-terminal domain directly contacts the C-terminal helicase domain, and the contact interface is a hotspot for disease-related mutations. Mutations at the interface destabilize Twinkle hexamer and reduce helicase activity. With HS-AFM, we observed that a highly dynamic Twinkle domain, which is likely to be the N-terminal domain, can protrude ∼5 nm to transiently capture nearby DNA and initialize Twinkle loading onto DNA. Moreover, structural analysis and subunit doping experiments suggest that Twinkle hydrolyzes ATP stochastically, which is distinct from related helicases from bacteriophages.

Extracellular proteolytic activation of Pseudomonas aeruginosa aminopeptidase (PaAP) and insight into the role of its non-catalytic N-terminal domain.

Pseudomonas aeruginosa secretes several endopeptidases, including elastase, alkaline proteinase (Apr), a lysine-specific endopeptidase (LysC), and an aminopeptidase (PaAP), all of which are important virulence factors. Activation of the endopeptidases requires removal of an inhibitory N-terminal propeptide. Activation of pro-PaAP, in contrast, requires C-terminal processing. The activating proteases of pro-PaAP and their cleavage site(s) have not yet been defined. Studying pro-PaAP processing in a wild type P. aeruginosa strain and strains lacking either elastase or both elastase and Apr, we detected three processing variants, each ~56 kDa in size (AP56). Activity assays and N- and C-terminal sequence analyses of these variants pointed at LysC as the principal activating protease, cleaving a Lys512-Ala513 peptide bond at the C-terminal end of pro-PaAP. Elastase and/or Apr are required for activation of LysC, suggesting both are indirectly involved in activation of PaAP. To shed light on the function(s) of the N-terminal domain of AP56, we purified recombinant AP56 and generated from it the 28 kDa catalytic domain (AP28). The kinetic constants (Km and Kcat) for hydrolysis of Leu-, Lys-, Arg- and Met-p-nitroanilide (pNA) derivatives by AP56 and AP28 were then determined. The catalytic coefficients (Kcat/Km) for hydrolysis of all four substrates by AP28 and AP56 were comparable, indicating that the non-catalytic domain is not involved in hydrolysis of small substrates. It may, however, regulate hydrolysis of natural peptides/proteins. Lys-pNA was hydrolyzed 2 to 3-fold more rapidly than Leu-pNA and ~8-fold faster than Arg- or Met-pNA, indicating that Lys-pNA was the preferred substrate.

Crystal Structure of a Soluble APOBEC3G Variant Suggests ssDNA to Bind in a Channel that Extends between the Two Domains

APOBEC3G (A3G) is a single-stranded DNA (ssDNA) cytosine deaminase that can restrict HIV-1 infection by mutating the viral genome. A3G consists of a non-catalytic N-terminal domain (NTD) and a catalytic C-terminal domain (CTD) connected by a short linker. While the CTD catalyzes cytosine deamination, the NTD is believed to provide additional affinity for ssDNA. Structures of both A3G domains have been solved individually; however, a full-length A3G structure has been challenging. Recently, crystal structures of full-length rhesus macaque A3G variants were solved which suggested dimerization mechanisms and RNA binding surfaces, whereas the dimerization appeared to compromise catalytic activity. We determined the crystal structure of a soluble variant of human A3G (sA3G) at 2.5 Å and from these data generated a model structure of wild-type A3G. This model demonstrated that the NTD was rotated 90° relative to the CTD along the major axis of the molecule, an orientation that forms a positively charged channel connected to the CTD catalytic site, consisting of NTD loop-1 and CTD loop-3. Structure-based mutations, in vitro deamination and DNA binding assays, and HIV-1 restriction assays identify R24, located in the NTD loop-1, as essential to a critical interaction with ssDNA. Furthermore, sA3G was shown to bind a deoxy-cytidine dinucleotide near the catalytic Zn2+, yet not in the catalytic position, where the interactions between deoxy-cytidines and CTD loop-1 and loop-7 residues were different from those formed with substrate. These new interactions suggest a mechanism explaining why A3G exhibits a 3′ to 5′ directional preference in processive deamination.

Two-Tier Screening Platform for Directed Evolution of Aminoacyl-tRNA Synthetases with Enhanced Stop Codon Suppression Efficiency.

Genetic code expansion through amber stop codon suppression provides a powerful tool for introducing non-proteinogenic functionalities into proteins for a broad range of applications. However, ribosomal incorporation of noncanonical amino acids (ncAAs) by means of engineered aminoacyl-tRNA synthetases (aaRSs) often proceeds with significantly reduced efficiency compared to sense codon translation. Here, we report the implementation of a versatile platform for the development of engineered aaRSs with enhanced efficiency in mediating ncAA incorporation by amber stop codon suppression. This system integrates a white/blue colony screen with a plate-based colorimetric assay, thereby combining high-throughput capabilities with reliable and quantitative measurement of aaRS-dependent ncAA incorporation efficiency. This two-tier functional screening system was successfully applied to obtain a pyrrolysyl-tRNA synthetase (PylRS) variant (CrtK-RS(4.1)) with significantly improved efficiency (+250-370 %) for mediating the incorporation of Nϵ -crotonyl-lysine and other lysine analogues of relevance for the study of protein post-translational modifications into a target protein. Interestingly, the beneficial mutations accumulated by CrtK-RS(4.1) were found to localize within the noncatalytic N-terminal domain of the enzyme and could be transferred to another PylRS variant, improving the ability of the variant to incorporate its corresponding ncAA substrate. This work introduces an efficient platform for the improvement of aaRSs that could be readily extended to other members of this enzyme family and/or other target ncAAs.

A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1

AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP hydrolysis in the AAA-domains is mediated by a non-catalytic N-terminal domain. The exact mechanisms that transmit the signal from the N-domain and coordinate the individual AAA-domains in the hexameric complex are still the topic of intensive research. Here, we present the characterization of a novel mutant variant of the eukaryotic AAA-ATPase Drg1 that shows dysregulation of ATPase activity and altered interaction with Rlp24, its substrate in ribosome biogenesis. This defective regulation is the consequence of amino acid exchanges at the interface between the regulatory N-domain and the adjacent D1 AAA-domain. The effects caused by these mutations strongly resemble those of pathological mutations of the AAA-ATPase p97 which cause the hereditary proteinopathy IBMPFD (inclusion body myopathy associated with Paget’s disease of the bone and frontotemporal dementia). Our results therefore suggest well conserved mechanisms of regulation between structurally, but not functionally related members of the AAA-family.

A non‐catalytic N‐terminal domain negatively influences the nucleotide exchange activity of translation elongation factor 1Bα

Eukaryotic translation elongation factor 1Bα (eEF1Bα) is a functional homolog of the bacterial factor EF-Ts, and is a component of the macromolecular eEF1B complex. eEF1Bα functions as a catalyst of guanine nucleotide exchange on translation elongation factor 1A (eEF1A). The C-terminal domain of eEF1Bα is necessary and sufficient for its catalytic activity, whereas the N-terminal domain interacts with eukaryotic translation elongation factor 1Bγ (eEF1Bγ) to form a tight complex. However, eEF1Bγ has been shown to enhance the catalytic activity of eEF1Bα attributed to the C-terminal domain of eEF1Bα. This suggests that the N-terminal domain of eEF1Bα may in some way influence the guanine nucleotide exchange process. We have shown that full-length recombinant eEF1Bα and its truncated forms are non-globular proteins with elongated shapes. Truncation of the N-terminal domain of eEF1Bα, which is dispensable for catalytic activity, resulted in acceleration of the rate of guanine nucleotide exchange on eEF1A compared to full-length eEF1Bα. A similar effect on the catalytic activity of eEF1Bα was observed after its interaction with eEF1Bγ. We suggest that the non-catalytic N-terminal domain of eEF1Bα may interfere with eEF1A binding to the C-terminal catalytic domain, resulting in a decrease in the overall rate of the guanine nucleotide exchange reaction. Formation of a tight complex between the eEF1Bγ and eEF1Bα N-terminal domains abolishes this inhibitory effect.

The non-catalytic N-terminal domain of ACS7 is involved in the post-translational regulation of this gene in Arabidopsis.

Post-transcriptional control of the expression of the 1-aminocyclopropane-1-carboxylate synthase (ACS) gene family is important for maintaining appropriate levels of ethylene production. However, the molecular mechanism underlying the post-transcriptional regulation of type 3 ACS proteins remains unclear. Multiple sequence alignment revealed that the N-terminus of type 3 ACSs was longer than that of other ACSs. Fusing the N-terminal 54 residues of ACS7, the sole type 3 ACS in Arabidopsis, to the β-glucuronidase (GUS) reporter significantly decreased the stability of N(7(1-54))-GUS fusion protein. Among these 54 residues, residues 1-14 conferred this negative effect on the GUS fusion gene. Consistently, a truncated form of ACS7 lacking residues 1-14 was more stable than full-length ACS7 when transgenically expressed in Arabidopsis and led to a more severe ethylene response phenotype in the light-grown transgenic seedlings. Interestingly, the ACS7 N-terminus had no effect on the stability of N(7)-GUS and ACS7 proteins at the etiolated seedling stage. Both exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) treatment and salt stress could rescue the levels of accumulation of N(7)-GUS fusion protein in light-grown seedlings. These results suggest that the non-catalytic N-terminus of ACS7 is involved in its own post-translational regulation. The proteasome inhibitor MG132 suppressed degradation of full-length ACS7 in vivo, but had little effect on the N-terminal truncated form of ACS7, indicating that the N-terminus mediates the regulation of ACS7 stability through the ubiquitin-26S proteasome pathway.

Binding of RNA by APOBEC3G controls deamination-independent restriction of retroviruses

APOBEC3G (A3G) is a host-encoded protein that potently restricts the infectivity of a broad range of retroviruses. This can occur by mechanisms dependent on catalytic activity, resulting in the mutagenic deamination of nascent viral cDNA, and/or by other means that are independent of its catalytic activity. It is not yet known to what extent deamination-independent processes contribute to the overall restriction, how they exactly work or how they are regulated. Here, we show that alanine substitution of either tryptophan 94 (W94A) or 127 (W127A) in the non-catalytic N-terminal domain of A3G severely impedes RNA binding and alleviates deamination-independent restriction while still maintaining DNA mutator activity. Substitution of both tryptophans (W94A/W127A) produces a more severe phenotype in which RNA binding and RNA-dependent protein oligomerization are completely abrogated. We further demonstrate that RNA binding is specifically required for crippling late reverse transcript accumulation, preventing proviral DNA integration and, consequently, restricting viral particle release. We did not find that deaminase activity made a significant contribution to the restriction of any of these processes. In summary, this work reveals that there is a direct correlation between A3G’s capacity to bind RNA and its ability to inhibit retroviral infectivity in a deamination-independent manner.

CDK-Dependent Potentiation of MPS1 Kinase Activity Is Essential to the Mitotic Checkpoint

Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached.

Phosphorylation Directly Regulates the Intrinsic DNA Cytidine Deaminase Activity of Activation-induced Deaminase and APOBEC3G Protein

The beneficial effects of DNA cytidine deamination by activation-induced deaminase (AID; antibody gene diversification) and APOBEC3G (retrovirus restriction) are tempered by probable contributions to carcinogenesis. Multiple regulatory mechanisms serve to minimize this detrimental outcome. Here, we show that phosphorylation of a conserved threonine attenuates the intrinsic activity of activation-induced deaminase (Thr-27) and APOBEC3G (Thr-218). Phospho-null alanine mutants maintain intrinsic DNA deaminase activity, whereas phospho-mimetic glutamate mutants are inactive. The phospho-mimetic variants fail to mediate isotype switching in activated mouse splenic B lymphocytes or suppress HIV-1 replication in human T cells. Our data combine to suggest a model in which this critical threonine acts as a phospho-switch that fine-tunes the adaptive and innate immune responses and helps protect mammalian genomic DNA from procarcinogenic lesions.

Regulation of yeast Yak1 kinase by PKA and autophosphorylation-dependent 14-3-3 binding

Yak1 is a member of an evolutionarily conserved family of Ser/Thr protein kinases known as dual-specificity Tyr phosphorylation-regulated kinases (DYRKs). Yak1 was originally identified as a growth antagonist, which functions downstream of Ras/PKA signalling pathway. It has been known that Yak1 is phosphorylated by PKA in vitro and is translocated to the nucleus upon nutrient deprivation. However, the regulatory mechanisms for Yak1 activity and localization are largely unknown. In the present study, we investigated the role of PKA and Bmh1, a yeast 14-3-3 protein, in regulation of Yak1. We demonstrate that PKA-dependent phosphorylation of Yak1 on Ser295 and two minor sites inhibits nuclear localization of Yak1. We also show that intramolecular autophosphorylation on at least four Ser/Thr residues in the non-catalytic N-terminal domain is required for full kinase activity of Yak1. The most potent autophosphorylation site, Thr335, plays an essential role for Bmh1 binding in collaboration with a yet unidentified second binding site in the N-terminal domain. Bmh1 binding decreases the catalytic activity of Yak1 without affecting its subcellular localization. Since the binding of 14-3-3 proteins to Yak1 coincides with PKA activity, such regulatory mechanisms might allow cytoplasmic retention of an inactive form of Yak1 under high glucose conditions.

Paralog specificity of mammalian SENPs for SUMO‐1 and SUMO‐2

There are four SUMO paralogs; SUMO‐2, ‐3 and ‐4 are closely related and about 40% identical to SUMO‐1. SENPs are SUMO specific proteases responsible for deSUMOylation. Using a panel of SUMO‐1 and ‐2 derived substrates and inhibitors we examined the distribution and paralog specificity of SENPs. The total deSUMOylation activity in tissues and mammalian cell lysates examined is 15‐fold higher using SUMO‐2 than SUMO‐1 aminomethylcoumarin (AMC). Mammalian cell lysates showed more reactivity with SUMO‐2 vinyl sulfone (VS) than with SUMO‐1VS. Only SENP1 reacts significantly with SUMO‐1VS. To confirm these observations we examined the paralog specificity of the recombinant catalytic domains (cSENPs). The cSENPs acted on SUMO‐2 with Km and Ki values from 50–200nM. cSENP1 and cSENP2 showed a 2–4 fold preference for SUMO‐1. All other cSENPs strongly preferred SUMO‐2. Because cSENP2 reacted with SUMO‐2 derivatives while full length SENP2 in lysates did not, we asked whether specificity was dependent on the N‐terminal non‐catalytic domain. Full length recombinant his6‐SENP1 showed the expected paralog specificity. In contrast the catalytic domain alone, his6‐SENP2 cleaved SUMO‐2‐AMC much faster than SUMO‐1‐AMC. Thus the non‐catalytic N‐terminal domain of SENP2 participates in determining paralog specificity. In summary, SUMO‐2/‐3 modification in vivo may play a more significant role than previously appreciated.

The SUMO-Specific Protease SENP5 Is Required for Cell Division

Posttranslational modification of substrates by the small ubiquitin-like modifier, SUMO, regulates diverse biological processes, including transcription, DNA repair, nucleocytoplasmic trafficking, and chromosome segregation. SUMOylation is reversible, and several mammalian homologs of the yeast SUMO-specific protease Ulp1, termed SENPs, have been identified. We demonstrate here that SENP5, a previously uncharacterized Ulp1 homolog, has SUMO C-terminal hydrolase and SUMO isopeptidase activities. In contrast to other SENPs, the C-terminal catalytic domain of SENP5 preferentially processed SUMO-3 compared to SUMO-1 precursors and preferentially removed SUMO-2 and SUMO-3 from SUMO-modified RanGAP1 in vitro. In cotransfection assays, SENP5 preferentially reduced high-molecular-weight conjugates of SUMO-2 compared to SUMO-1 in vivo. Full-length SENP5 localized to the nucleolus. Deletion of the noncatalytic N-terminal domain led to loss of nucleolar localization and increased de-SUMOylation activity in vivo. Knockdown of SENP5 by RNA interference resulted in increased levels of SUMO-1 and SUMO-2/3 conjugates, inhibition of cell proliferation, defects in nuclear morphology, and appearance of binucleate cells, revealing an essential role for SENP5 in mitosis and/or cytokinesis. These findings establish SENP5 as a SUMO-specific protease required for cell division and suggest that mechanisms involving both the catalytic and noncatalytic domains determine the distinct substrate specificities of the mammalian SUMO-specific proteases.

Intramolecular Dephosphorylation of ERK by MKP3

The dual specificity mitogen-activated protein kinase phosphatase MKP3 downregulates mitogenic signaling through dephosphorylation of extracellular signal-regulated kinase (ERK). Like other MKPs, MKP3 consists of a noncatalytic N-terminal domain and a catalytic C-terminal domain. ERK binding to the N-terminal noncatalytic domain of MKP3 has been shown to increase (up to 100-fold) the catalytic activity of MKP3 toward small artificial substrates. Here, we address the function of the N-terminal domain of MKP3 in either inter- or intramolecular dephosphorylation of pERK (phosphorylated ERK) and the stoichiometry of the MKP3/pERK Michaelis complex. These are important mechanistic distinctions given the observation that ERK exists in a monomer/dimer equilibrium that is shifted toward the dimer when phosphorylated and given that MKP3 undergoes catalytic activation toward other substrates when bound to ERK. Wild-type and engineered mutants of ERK and MKP3, binding analyses, reaction kinetics, and chemical cross-linking studies were used to demonstrate that the monomer of MKP3 binds to the monomeric form of pERK and that MKP3 within the resulting heterodimer performs intramolecular dephosphorylation of pERK. This study provides the first direct evidence that MKP3 utilizes intramolecular dephosphorylation between a complex consisting of one molecule each of MKP3 and ERK. Catalytic activation and substrate tethering by MKP3 lead to a >or=4000-fold rate enhancement (k(cat)/K(m)) for dephosphorylation of pERK.

Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A

Protein kinases of the DYRK (‘dual-specificity tyrosine-regulated kinase’) family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319–Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.

Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A.

Protein kinases of the DYRK ('dual-specificity tyrosine-regulated kinase') family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319-Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.

Caspase-mediated Cleavage of the Ca2+/Calmodulin-dependent Protein Kinase-like Kinase Facilitates Neuronal Apoptosis

This study was designed to identify the role of a recently identified Ca(2+)/calmodulin-dependent protein kinase (CaMK)-like kinase (CaMKLK) in neuronal apoptosis. For this purpose, we studied proteolytic cleavage of CaMKLK by caspases in vitro and in neuronal NG108 cells. In addition, we have investigated the effect of overexpression of wild type and mutant CaMKLK proteins on staurosporine- and serum deprivation-induced apoptosis of NG108 cells. We found that CaMKLK is a substrate for caspase-3 and -8, both in vitro and in NG108 cells during staurosporine- and serum withdrawal-induced apoptosis. Substitution of an aspartic acid residue at position 62 in an asparagine residue within a putative caspase cleavage site completely blocked cleavage of CaMKLK, strongly indicating that (59)DEND(62) is the caspase recognition site. Overexpression of an Asp(62) --> Asn CaMKLK mutant protected NG108 cells from staurosporine-induced apoptosis to a similar extent as Bcl-x(L). In contrast, overexpression of wild type CaMKLK did not lead to protection. Moreover, microinjection of Asp(62) --> Asn CaMKLK protected NG108 cells from serum deprivation-induced apoptosis, while overexpression of a caspase-generated noncatalytic N-terminal CaMKLK fragment exacerbated apoptosis. Together, our data suggest that cleavage of CaMKLK and generation of the noncatalytic N-terminal domain of CaMKLK facilitate neuronal apoptosis.