Substrate specificities and activities of AZAP family Arf GAPs in vivo

The ADP-ribosylation factor (Arf) GTPases are important regulators of vesicular transport in eukaryotic cells. Like other GTPases, the Arfs require guanine nucleotide exchange factors to facilitate GTP loading and GTPase-activating proteins (GAPs) to promote GTP hydrolysis. Whereas there are only six mammalian Arfs, the human genome encodes over 20 proteins containing Arf GAP domains. A subset of these, referred to as AZAPs (Randazzo PA, Hirsch DS. Cell Signal 16: 401-413, 2004), are characterized by the presence of at least one NH(2)-terminal pleckstrin homology domain and two or more ankyrin repeats following the GAP domain. The substrate specificities of these proteins have been previously characterized by using in vitro assay systems. However, a limitation of such assays is that they may not accurately represent intracellular conditions, including posttranslational modifications, or subcellular compartmentalization. Here we present a systematic analysis of the GAP activity of seven AZAPs in vivo, using an assay for measurement of cellular Arf-GTP (Santy LC, Casanova JE. J Cell Biol 154: 599-610, 2001). In agreement with previous in vitro results, we found that ACAP1 and ACAP2 have robust, constitutive Arf6 GAP activity in vivo, with little activity toward Arf1. In contrast, although ARAP1 was initially reported to be an Arf1 GAP, we found that it acts primarily on Arf6 in vivo. Moreover, this activity appears to be regulated through a mechanism involving the NH(2)-terminal sterile-alpha motif. AGAP1 is unique among the AZAPs in its specificity for Arf1, and this activity is dependent on its NH(2)-terminal GTPase-like domain. Finally, we found that expression of AGAP1 induces a surprising reciprocal activation of Arf6, which suggests that regulatory cross talk exists among Arf isoforms.     

Substrate specificities of family 1 UGTs gained by domain swapping

Family 1 glycosyltransferases are a group of enzymes known to embrace a large range of different substrates. This study devises a method to enhance the range of substrates even further by combining domains from different glycosyltransferases to gain improved substrate specificity and catalytic efficiency. Chimeric glycosyltransferases were made by combining domains from seven different family 1 glycosyltransferases, UGT71C1, UGT71C2, UGT71E1, UGT85C1, UGT85B1, UGT88B1 and UGT94B1. Twenty different chimeric glycosyltransferases were formed of which twelve were shown to be catalytically active. The chimeric enzymes of Arabidopsis thaliana UGT71C1 and UGT71C2 showed major changes in acceptor substrate specificity and were able to glycosylate etoposide significantly better than the parental UGT71C1 and UGT71C2 enzymes, with K_cat and efficiency coefficients 3.0 and 2.6 times higher, respectively. Chimeric glycosyltransferases of UGT71C1 combined with Stevia rebaudiana UGT71E1, also afforded enzymes with high catalytic efficiency, even though the two enzymes only display 38% amino acid sequence identity. These chimeras show a significantly altered regiospecificity towards especially trans-resveratrol, enabling the production of trans-resveratrol-β-4′-O-glucoside (resveratroloside). The study demonstrates that it is possible to obtain improved catalytic properties by combining domains from both closely as well as more distantly related glycosyltransferases. The substrate specificity gained by the chimeras is difficult to predict because factors determining the acceptor specificity reside in the N- terminal as well as the C-terminal domains.     

Substrate specificities of wild and mutated farnesyl diphosphate synthases from Bacillus stearothermophilus with artificial substrates

To determine the substrate specificities of wild and mutated types of farnesyl diphosphate (FPP) synthases from Bacillus stearothermophilus, we examined the reactivities of 8-hydroxygeranyl diphosphate (HOGPP) and 8-methoxygeranyl diphosphate (CH(3)OGPP) as allylic substrate homologs. The wild-type FPP synthase reaction of HOGPP (and CH(3)OGPP) with isopentenyl diphosphate (IPP) gave hydroxyfarnesyl- (and methoxyfarnesyl-) diphosphates that stopped at the first stage of condensation. On the other hand, with mutated type FPP synthase (Y81S), the former gave hydroxygeranylgeranyl diphosphate as the main double-condensation product together with hydroxyfarnesyl diphosphate as a single-condensation product and a small amount of hydroxygeranylfarnesyl diphosphate as a triple-condensation product. Moreover, the latter gave a double-condensation product, methoxygeranylgeranyl diphosphate, as the main product and only a trace of methoxyfarnesyl diphosphate was obtained.     

Determination of substrate specificity and putative substrates of Chk2 kinase

Chk2/hCds1, the human homolog of Saccharomyces cerevisiae Rad53p and Schizosaccharomyces pombe Cds1p, plays a critical role in the DNA damage checkpoint pathway. While several in vivo targets of Chk2 have been identified, the other target proteins of Chk2 responsible for multiple functions, such as cell cycle arrest, DNA repair, and apoptosis, remain to be elucidated. We utilized the GST-peptide approach to identify physiological substrates for Chk2. Mutational analyses using GST-linked Cdc25A containing serine 123 revealed that residues at positions -5 and -3 are critical determinants for the recognition of the Chk2 substrate. We determined the general phosphorylation consensus sequence and identified in vitro targets of Chk2 using GST peptides as substrates. The newly identified in vitro target proteins include Abl1, Bub1R, Bub1, Bub3, Psk-H1, Smc3, Plk1, Cdc25B, Dcamkl1, Mre11, Pms1, and Xrcc9.     

Substrate specificities of tobacco chitinases

Ten tobacco chitinases (1,4-N-acetyl-β-D-glucosaminide glycanhydrolase, EC 3.2.1.14) were purified from tobacco leaves hypersensitively reacting to tobacco mosaic virus. The 10 enzymes, which belong to five distinct structural classes of plant chitinases, were incubated with several potential substrates such as chitin, a β-1,4 N-acetyl-D-glucosamine (GlcNAc) polymer, chitosan (partially deacetylated chitin), chitin oligomers of variable length and bacterial cell wall. Tobacco chitinases are all endo-type enzymes that liberate oligomers from chitin and are capable of processing the chito-oligomers further at differential rates. Chitin reaction products were separated and quantified by HPLC and differential kinetics of oligomer accumulation and degradation were observed with the distinct classes of chitinases. Depending on the substrate to be hydrolysed, each isoform displayed a different spectrum of activity. For example, class I isoforms were the most active on chitin and (GlcNAc)_4–6 whereas class III basic isoforms were the most efficient in inducing bacterial lysis. Class V and class VI chitinases were shown to more readily hydrolyse chitin oligomers than the chitin polymer itself. Together, these data indicate that the 10 tobacco chitinases represent complementary enzymes which may have synergistic effects on their substrates. This paper discusses their implication in plant defense by attacking pathogen's structural components and in plant development by maturing signal molecules.     

Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment

Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti(4+)-immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO(2) enrichment method. We also examined the effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improve the enrichment of phosphopeptides containing multiple basic residues. We found that Ti(4+)-IMAC in combination with TFA in the loading buffer, outperformed all other methods tested, enabling the identification of around 5000 unique phosphopeptides containing multiple basic residues from 400 μg of a HeLa cell lysate digest. In comparison, ～ 2000 unique phosphopeptides could be identified by Ti(4+)-IMAC with HFP and close to 3000 by TiO(2). We confirmed, by motif analysis, the basic phosphopeptides enrich the number of putative basophilic kinases substrates. In addition, we performed an experiment using the SCX/Ti(4+)-IMAC methodology alongside the use of collision-induced dissociation (CID), higher energy collision induced dissociation (HCD) and electron transfer dissociation with supplementary activation (ETD) on considerably more complex sample, consisting of a total of 400 μg of triple dimethyl labeled MCF-7 digest. This analysis led to the identification of over 9,000 unique phosphorylation sites. The use of three peptide activation methods confirmed that ETD is best capable of sequencing multiply charged peptides. Collectively, our data show that the combination of SCX and Ti(4+)-IMAC is particularly advantageous for phosphopeptides with multiple basic residues.     

Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family

The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74‐compound library of known Nudix enzyme substrates. We found substrates for four enzymes with k_cat/K_m values >10,000 M^?1 s^?1: Q92EH0_LISIN of Listeria innocua serovar 6a against ADP‐ribose, Q5LBB1_BACFN of Bacillus fragilis against 5‐Me‐CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8‐oxo‐dATP and 3'‐dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty‐two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported k_cat/K_m values exhibited against these canonical substrates are well under 10⁵ M^?1 s^?1. By contrast, several Nudix enzymes show much larger k_cat/K_m values (in the range of 10⁵ to >10⁷ M^?1 s^?1) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810–1822. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Isolation of two members of the rat MAP kinase kinase gene family

Mitogen-activated protein (MAP) kinase kinase (MAPKK) is a recently characterized activator of MAP kinase (MAPK), and is considered to be regulated by a protooncogene product c-Raf-1. It is, however, unclear whether the signals originating from c-Raf-1 utilize this phosphorylation cascade to lead to oncogenesis. To clarify this point, we isolated rat MAPKK cDNAs, and identified two distinct cDNAs encoding MAPKK and a highly related kinase, both with molecular weights of 5 kDa (MEK1 and MEK2). Genomic Southern blot analyses suggested that MAPKK. may form a large gene family.     

Differing substrate specificities of members of the DYRK family of arginine-directed protein kinases.

The mammalian DYRK (dual specificity tyrosine phosphorylated and regulated kinase) family of protein kinases comprises a number of related, but poorly understood enzymes. DYRK1A is nuclear while DYRKs 2 and 3 are cytoplasmic. We recently showed that DYRK2 phosphorylates the translation initiation factor eIF2B at Ser539 in its epsilon-subunit and thereby "primes" its phosphorylation by glycogen synthase kinase-3. Here we have used peptides based on the sequence around Ser539 to help define the specificity of DYRK2/3 in comparison with DYRK1A. These kinases require an arginine N-terminal to the target residue for efficient substrate phosphorylation. This cannot be replaced even by lysine. A peptide with arginine at -2 is phosphorylated much less well by all three kinases than one with arginine at -3. Replacement of the +1 proline by alanine almost completely eliminates substrate phosphorylation, but valine here does allow phosphorylation especially by DYRK2. This study reveals both similarities and differences in the specificities of these arginine-dependent protein kinases.     

QIKS – Quantitative identification of kinase substrates

Signaling networks regulate cellular responses to external stimuli through post‐translational modifications such as protein phosphorylation. Phosphoproteomics facilitate the large‐scale identification of kinase substrates. Yet, the characterization of critical connections within these networks and the identification of respective kinases remain the major analytical challenge. To address this problem, we present a novel approach for the identification of direct kinase substrates using chemical genetics in combination with quantitative phosphoproteomics. Quantitative identification of kinase substrates (QIKS) is a novel‐screening platform developed for the proteome‐wide substrate‐analysis of specific kinases. Here, we aimed to identify substrates of mitogen‐activated protein kinase/Erk kinase (Mek1), an essential kinase in the mitogen‐activated protein kinase cascade. An ATP analog‐sensitive mutant of Mek1 (Mek1‐as) was incubated with a cell extract from Mek1 deficient cells. Phosphorylated proteins were analyzed by LC‐MS/MS of IMAC‐enriched phosphopeptides, labeled differentially for relative quantification. The identification of extracellular regulated kinase 1/2 as the sole cytoplasmic substrates of MEK1 validates the applicability of this approach and suggests that QIKS could be used to identify substrates of a wide variety of kinases.     

A family of polypeptide substrates and inhibitors of insulin receptor kinase

Previous studies have shown that reduced carbamoylmethylated lysozyme (RCAM-lysozyme, MW approximately 14.5K) is a substrate and inhibitor (Ki approximately 0.6 microM) of insulin receptor kinase (InsRK) autophosphorylation (Kohanski & Lane, 1986; Lane & Kohanski, 1986). In this study we have prepared a family of defined modified derivatives of RCAM-lysozyme and used them to probe the nature of the substrate and inhibitory sites of InsRK. All open-chain derivatives of lysozyme in which either the tryptophanyl, methionyl, cysteinyl, arginyl, or histidyl side chains were modified served as substrates and were potent inhibitors of InsRK autophosphorylation. This was true whether the substitutions were either hydrophilic or hydrophobic, although the hydrophilic derivatives had a higher inhibitory potency. Tryptic peptides derived from RCAM-lysozyme, however, were inactive as inhibitors, and a mixture of the three cyanogen bromide fragments (containing 12, 24, and 93 amino acids, respectively) was found to be less potent in inhibiting the receptor kinase. Derivatization of either tyrosyl or carboxyl side chains produced derivatives that were neither substrates nor capable of inhibiting receptor autophosphorylation. Derivatives with modified amino groups were substrates for InsRK but were not able to inhibit InsRK autophosphorylation. The present study suggests that (a) unphosphorylated InsRK has a large hydrophilic substrate binding domain and is effectively inhibited by long-chain polypeptides but not by short sequences, (b) some of the amino, carboxyl, and hydroxyphenyl side chains are essential to the inhibitory nature of these polypeptides, and (c) derivatives that fail to inhibit autophosphorylation can still be recognized and phosphorylated by active InsRK.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate specificities of bacterial and human AlkB proteins

Methylating agents introduce cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues into nucleic acids, and it was recently demonstrated that the Escherichia coli AlkB protein and two human homologues, hABH2 and hABH3, can remove these lesions from DNA by oxidative demethylation. Moreover, AlkB and hABH3 were also found to remove 1-meA and 3-meC from RNA, suggesting that cellular RNA repair can occur. We have here studied the preference of AlkB, hABH2 and hABH3 for single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), and show that AlkB and hABH3 prefer ssDNA, while hABH2 prefers dsDNA. This was consistently observed with three different oligonucleotide substrates, implying that the specificity for single-stranded versus double-stranded DNA is sequence independent. The dsDNA preference of hABH2 was observed only in the presence of magnesium. The activity of the enzymes on single-stranded RNA (ssRNA), double-stranded RNA (dsRNA) and DNA/RNA hybrids was also investigated, and the results generally confirm the notion that while AlkB and hABH3 tend to prefer single-stranded nucleic acids, hABH2 is more active on double-stranded substrates. These results may contribute to identifying the main substrates of bacterial and human AlkB proteins in vivo.     

Evidence for distinct substrate specificities of importin alpha family members in nuclear protein import.

Importin alpha plays a pivotal role in the classical nuclear protein import pathway. Importin alpha shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin beta-dependent import pathway. In contrast to what is found for importin beta, several isoforms of importin alpha, which can be grouped into three subfamilies, exist in higher eucaryotes. We describe here a novel member of the human family, importin alpha7. To analyze specific functions of the distinct importin alpha proteins, we recombinantly expressed and purified five human importin alpha's along with importin alpha from Xenopus and Saccharomyces cerevisiae. Binding affinity studies showed that all importin alpha proteins from humans or Xenopus bind their import receptor (importin beta) and their export receptor (CAS) with only marginal differences. Using an in vitro import assay based on permeabilized HeLa cells, we compared the import substrate specificities of the various importin alpha proteins. When the substrates were tested singly, only the import of RCC1 showed a strong preference for one family member, importin alpha3, whereas most of the other substrates were imported by all importin alpha proteins with similar efficiencies. However, strikingly different substrate preferences of the various importin alpha proteins were revealed when two substrates were offered simultaneously.     

A putative adenosine kinase family protein possesses adenosine diphosphatase activity

Adenosine kinase is a potential target for development of new types of drugs. The COG1839 family has been defined as “adenosine-specific kinase” family based on structural analysis and the adenosine-binding ability of a family member, PAE2307. However, there has been no experimental evidence with regard to the enzymatic function of this protein family. Here we measured the enzymatic activity of TTHA1091, a COG1839 family protein from Thermus thermophilus HB8. The phosphorylation of adenosine by TTHA1091 was undetectable when ATP or ADP were used as phosphate donor. However, the degradation of ADP to AMP was detected, indicating that this protein possessed adenosine diphosphatase (ADPase) activity. The (ADPase) activity was inhibited by divalent cations and was specific to ADP and CDP. Thus, this study provides the first experimental evidence for the enzymatic function of the “adenosine-specific kinase” family and suggests a need to reexamine its functional annotation.     

Erythropoietin and interleukin-2 activate distinct JAK kinase family members.

The erythropoietin (EPO) receptor and the interleukin-2 (IL-2) receptor beta-chain subunit are members of the cytokine receptor superfamily. They have conserved primary amino acid sequences in their cytoplasmic domains and activate phosphorylation of common substrates, suggesting common biochemical signaling mechanisms. We have generated a cell line, CTLL-EPO-R, that contains functional cell surface receptors for both EPO and IL-2. CTLL-EPO-R cells demonstrated similar growth kinetics in EPO and IL-2. Stimulation with EPO resulted in the rapid, dose-dependent tyrosine phosphorylation of JAK2. In contrast, stimulation with IL-2 or the related cytokine IL-4 resulted in the rapid, dose-dependent tyrosine phosphorylation of JAK1 and an additional 116-kDa protein. This 116-kDa protein was itself immunoreactive with a polyclonal antiserum raised against JAK2 and appears to be a novel member of the JAK kinase family. Immune complex kinase assays confirmed that IL-2 and IL-4 activated JAK1 and EPO activated JAK2. These results demonstrate that multiple biochemical pathways are capable of conferring a mitogenic signal in CTLL-EPO-R cells and that the EPO and IL-2 receptors interact with distinct JAK kinase family members within the same cellular background.