A redox‐linked novel pathway for arsenic‐mediated RET tyrosine kinase activation

We examined the biochemical effects of arsenic on the activities of RET proto‐oncogene (c‐RET protein tyrosine kinases) and RET oncogene (RET‐MEN2A and RET‐PTC1 protein tyrosine kinases) products. Arsenic activated c‐RET kinase with promotion of disulfide bond‐mediated dimerization of c‐RET protein. Arsenic further activated RET‐MEN2A kinase, which was already 3‐ to 10‐fold augmented by genetic mutation compared with c‐RET kinase activity, with promotion of disulfide bond‐mediated dimerization of RET‐MEN2A protein (superactivation). Arsenic also increased extracellular domain‐deleted RET‐PTC1 kinase activity with promotion of disulfide bond‐mediated dimerization of RET‐PTC1 protein. Arsenic increased RET‐PTC1 kinase activity with cysteine 365 (C365) replaced by alanine with promotion of dimer formation but not with cysteine 376 (C376) replaced by alanine. Our results suggest that arsenic‐mediated regulation of RET kinase activity is dependent on conformational change of RET protein through modulation of a special cysteine sited at the intracellular domain in RET protein (relevant cysteine of C376 in RET‐PTC1 protein). Moreover, arsenic enhanced the activity of immunoprecipitated RET protein with increase in thiol‐dependent dimer formation. As arsenic (14.2 µM) was detected in the cells cultured with arsenic (100 µM), direct association between arsenic and RET in the cells might modulate dimer formation. Thus, we demonstrated a novel redox‐linked mechanism of activation of arsenic‐mediated RET proto‐oncogene and oncogene products. J. Cell. Biochem. 110: 399–407, 2010. © 2010 Wiley‐Liss, Inc.     

Expression and regulation of mutant forms of cardiac TnI in a reconstituted actomyosin system: Role of kinase dependent phosphorylation

When phosphorylated, the inhibitory subunit of troponin (TnI) causes a loss in calcium sensitivity and a decrease in actomyosin ATPase. To examine this process, we bacterially expressed wild type TnI and TnI mutants in which serine 22 and 23, a putative protein kinase A (PKA) site, and threonine 143, a putative protein kinase C (PKC) site, were replaced by alanine S22A/23A and T143A. PKA dependent phosphorylation was ~90% reduced in the S22A/23A mutant and unaffected in T143A. PKC dependent phosphorylation was markedly reduced in T143A relative both to a wild type construct and to S22A/23A, although some residual phosphorylation (likely at sites other than T143) was seen. The calcium sensitivity (i.e. inhibition of actomyosin ATPase in the presence of EGTA) and regulation of the reconstituted actomyosin system was preserved in the absence of phosphorylation using wild type TnI or either mutant. Calcium sensitivity was decreased by both PKA and PKC with the wild type TnI but was unaffected by PKA when the S22A/23A mutant was employed and by PKC when the T143A mutant was reconstituted. The calcium dependency of the ATPase curve was substantially right shifted when PKC phosphorylated wild type TnI was employed for regulation, and this was markedly attenuated when T143 A was reassociated (although a slight rightward shift and a reduction in maximal ATPase activity was still seen). These data confirm that phosphorylation of TnI by regulatory kinases plays a major role in the regulation of myofibrillar ATPase. The N-terminal serines (22 and 23) appear to be uniquely important for the PKA response whereas threonine 143 is involved in the PKC response although other residues may also have functional significance.     

Characterization of inhibitory signaling motifs of the natural killer cell receptor Siglec-7: attenuated recruitment of phosphatases by the receptor is attributed to two amino acids in the motifs

Siglec-7 (p75/AIRM1) is an inhibitory receptor on human natural killer cells (NK cells) and monocytes. The cytoplasmic domain of Siglec-7 contains two signaling motifs: a membrane-proximal immunoreceptor tyrosine-based inhibitory motif (ITIM) (Ile435-Gln-Tyr-Ala-Pro-Leu440) and a membrane-distal motif (Asn458-Glu-Tyr-Ser-Glu-Ile463). We report here that, upon pervanadate (PV) treatment, Siglec-7 recruited the protein tyrosine phosphatases Src homology-2 (SH2) domain-containing protein-tyrosine phosphatase-1 (SHP-1) and SHP-2 less efficiently than did other inhibitory receptors such as Siglec-9 and leukocyte-associated Ig-like receptor (LAIR-1). Alignment of the amino acid sequences of the two Siglecs revealed only three amino acids difference in these motifs. To identify the amino acid(s) critical to recruitment efficiency, we prepared a series of Siglec-7-based mutants in which each of the three amino acids were replaced with the corresponding one of Siglec-9 (I435L, P439S, and N458T mutants). P439S and N458T mutants showed pronounced enhancement of SHP recruitment, but I435L mutant had little effect. A double mutant (P439S, N458T) or triple mutant (I435L, P439S, N458T) recruited SHPs as much as did Siglec-9, indicating that Pro439 in the proximal motif and Asn458 in the distal motif of Siglec-7 attenuate its ability to recruit phosphatases. These amino acids appeared to affect not only phosphatase recruitment but also the subsequent attenuation of Syk phosphorylation.     

Quantification of the Dynamic Phosphorylation Process of ERK Using Stable Isotope Dilution Selective Reaction Monitoring Mass Spectrometry

Mitogen‐activated protein (MAP) kinase signaling is critical for various cellular responses, including cell proliferation, differentiation, and cell death. The MAP kinase cascade is conserved in the eukaryotic kingdom as a three‐tiered kinase module—MAP kinase kinase kinase, MAP kinase kinase, and MAP kinase—that transduces signals via sequential phosphorylation upon stimulation. Dual phosphorylation of MAP kinase on the conserved threonine‐glutamic acid‐tyrosine (TEY) motif is essential for its catalytic activity and signal activation; however, the molecular mechanism by which the two residues are phosphorylated remains elusive. In the present study, the pattern of dual phosphorylation of extracellular signal‐regulated kinase (ERK) is profiled on the TEY motif using stable isotope dilution (SID)‐selective reaction monitoring (SRM) mass spectrometry (MS) to elucidate the order and magnitude of endogenous ERK phosphorylation in cellular model systems. The SID‐SRM‐MS analysis of phosphopeptides demonstrates that tyrosine phosphorylation in the TEY motif is dynamic, while threonine phosphorylation is static. Analyses of the mono‐phosphorylatable mutants ERK^T202A and ERK^Y204F indicate that phosphorylation of tyrosine is not affected by the phosphorylation state of threonine, while threonine phosphorylation depends on tyrosine phosphorylation. The data suggest that dual phosphorylation of ERK is a highly ordered and restricted mechanism determined by tyrosine phosphorylation.     

Mechanisms of checkpoint kinase Rad53 inactivation after a double-strand break in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, double-strand breaks (DSBs) activate DNA checkpoint pathways that trigger several responses including a strong G(2)/M arrest. We have previously provided evidence that the phosphatases Ptc2 and Ptc3 of the protein phosphatase 2C type are required for DNA checkpoint inactivation after a DSB and probably dephosphorylate the checkpoint kinase Rad53. In this article we have investigated further the interactions between Ptc2 and Rad53. We showed that forkhead-associated domain 1 (FHA1) of Rad53 interacts with a specific threonine of Ptc2, T376, located outside its catalytic domain in a TXXD motif which constitutes an optimal FHA1 binding sequence in vitro. Mutating T376 abolishes Ptc2 interaction with the Rad53 FHA1 domain and results in adaptation and recovery defects following a DSB. We found that Ckb1 and Ckb2, the regulatory subunits of the protein kinase CK2, are necessary for the in vivo interaction between Ptc2 and the Rad53 FHA1 domain, that Ckb1 binds Ptc2 in vitro and that ckb1Delta and ckb2Delta mutants are defective in adaptation and recovery after a DSB. Our data thus strongly suggest that CK2 is the kinase responsible for the in vivo phosphorylation of Ptc2 T376.     

The crystal structure of TrxA(CACA): Insights into the formation of a [2Fe-2S] iron-sulfur cluster in an Escherichia coli thioredoxin mutant

Escherichia coli thioredoxin is a small monomeric protein that reduces disulfide bonds in cytoplasmic proteins. Two cysteine residues present in a conserved CGPC motif are essential for this activity. Recently, we identified mutations of this motif that changed thioredoxin into a homodimer bridged by a [2Fe-2S] iron-sulfur cluster. When exported to the periplasm, these thioredoxin mutants could restore disulfide bond formation in strains lacking the entire periplasmic oxidative pathway. Essential for the assembly of the iron-sulfur was an additional cysteine that replaced the proline at position three of the CGPC motif. We solved the crystalline structure at 2.3 Angstroms for one of these variants, TrxA(CACA). The mutant protein crystallized as a dimer in which the iron-sulfur cluster is replaced by two intermolecular disulfide bonds. The catalytic site, which forms the dimer interface, crystallized in two different conformations. In one of them, the replacement of the CGPC motif by CACA has a dramatic effect on the structure and causes the unraveling of an extended alpha-helix. In both conformations, the second cysteine residue of the CACA motif is surface-exposed, which contrasts with wildtype thioredoxin where the second cysteine of the CXXC motif is buried. This exposure of a pair of vicinal cysteine residues apparently allows thioredoxin to acquire an iron-sulfur cofactor at its active site, and thus a new activity and mechanism of action.     

Mutations in Critical Domains Confer the Human mTOR Gene Strong Tumorigenicity

Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that regulates cell growth, proliferation, and survival. mTOR is frequently activated in human cancers and is a commonly sought anticancer therapeutic target. However, whether the human mTOR gene itself is a proto-oncogene possessing tumorigenicity has not been firmly established. To answer this question, we mutated evolutionarily conserved amino acids, generated eight mutants in the HEAT repeats (M938T) and the FAT (W1456R and G1479N) and kinase (P2273S, V2284M, V2291I, T2294I, and E2288K) domains of mTOR, and studied their oncogenicity. On transient expression in HEK293T cells, these mTOR mutants displayed elevated protein kinase activities accompanied by activated mTOR/p70S6K signaling at varying levels, demonstrating the gain of function of the mTOR gene with these mutations. We selected P2273S and E2288K, the two most catalytically active mutants, to further examine their oncogenicity and tumorigenicity. Stable expression of the two mTOR mutants in NIH3T3 cells strongly activated mTOR/p70S6K signaling, induced cell transformation and invasion, and remarkably, caused rapid tumor formation and growth in athymic nude mice after subcutaneous inoculation of the transfected cells. This study confirms the oncogenic potential of mTOR suggested previously and demonstrates for the first time its tumorigenicity. Thus, beyond the pivotal position of mTOR to relay the oncogenic signals from the upstream phosphatidylinositol 3-kinase/Akt pathway in human cancer, mTOR is capable potentially of playing a direct role in human tumorigenesis if mutated. These results also further support the conclusion that mTOR is a major therapeutic target in human cancers.     

Role of threonine residues in the regulation of manganese-dependent arabidopsis serine/threonine/tyrosine protein kinase activity

Tyrosine phosphorylation in plants could be performed only by dual-specificity kinases. Arabidopsis thaliana dual-specificity protein kinase (AtSTYPK) exhibited strong preference for manganese over magnesium for its kinase activity. The kinase autophosphorylated on serine, threonine and tyrosine residues and phosphorylated myelin basic protein on threonine and tyrosine residues. The AtSTYPK harbors manganese dependent serine/threonine kinase domain, COG3642. His248 and Ser265 on COG3642 are conserved in AtSTYPK and the site-directed mutant, H248A showed loss of serine/threonine kinase activity. The protein kinase activity was abolished when Thr208 in the TEY motif and Thr293 of the activation loop were converted to alanine. The conversion of Thr284 in the activation loop to alanine resulted in an increased phosphorylation. This study reports the first identification of a manganese dependent dual-specificity kinase and the importance of Thr208, Thr284, and Thr293 residues in the regulation of kinase activity.     

Impact of cysteine variants on the structure,activity, and stability of recombinant human α-galactosidase A

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity.     

Site-directed chemical labeling of extracellular loops in a membrane protein. The topology of the Na,K-ATPase alpha-subunit

We have mapped the membrane topology of the renal Na,K-ATPase alpha-subunit by using a combination of introduced cysteine mutants and surface labeling with a membrane impermeable Cys-directed reagent, N-biotinylaminoethyl methanethiosulfonate. To begin our investigation, two cysteine residues (Cys(911) and Cys(964)) in the wild-type alpha-subunit were substituted to create a background mutant devoid of exposed cysteines (Lutsenko, S., Daoud, S., and Kaplan, J. H. (1997) J. Biol. Chem. 272, 5249-5255). Into this background construct were then introduced single cysteines in each of the five putative extracellular loops (P118C, T309C, L793C, L876C, and M973C) and the resulting alpha-subunit mutants were co-expressed with the beta-subunit in baculovirus-infected insect cells. All of our expressed Na,K-ATPase mutants were functionally active. Their ATPase, phosphorylation, and ouabain binding activities were measured, and the turnover of the phosphoenzyme intermediate was close to the wild-type enzyme, suggesting that they are folded properly in the infected cells. Incubation of the insect cells with the cysteine-selective reagent revealed essentially no labeling of the alpha-subunit of the background construct and labeling of all five mutants with single cysteine residues in putative extracellular loops. Two additional mutants, V969C and L976C, were created to further define the M9M10 loop. The lack of labeling for these two mutants showed that although Met(973) is apparently exposed, Val(969) and Leu(976) are not, demonstrating that this method may also be utilized to define membrane aqueous boundaries of membrane proteins. Our labeling studies are consistent with a specific 10-transmembrane segment model of the Na,K-ATPase alpha-subunit. This strategy utilized only functional Na,K-ATPase mutants to establish the membrane topology of the entire alpha-subunit, in contrast to most previously applied methods.     

Biphasic activation of two mitogen-activated protein kinases during the cell cycle in mammalian cells.

We studied mitogen-activated protein kinase (MAPK) activities during the cell cycle of Chinese hamster ovary (CHO) cells using site-specific antibodies against extracellular signal-regulated kinase-1, a 44-kDa MAPK (Boulton, T.G., Yancopoulos, G.D., Gregory, J.S., Slauer, C., Moomaw, C., Hsu, J., and Cobb, M.H. (1990) Science 249, 64-67). These antibodies detected two distinct MAPKs (44- and 42-kDa MAPKs) in CHO cells. CHO cells were arrested at metaphase in the M phase by treatment with nocodazole, and activities of MAPKs were analyzed at specific time points after release from arrest. Immune complex kinase assay and renaturation and phosphorylation assay in substrate-containing gel revealed that both 44- and 42-kDa MAPKs had activities in the G1 through S and G2/M phases and were activated biphasically, in the G1 phase and around the M phase. MAPKs were inactivated in metaphase-arrested cells. The amount of MAPKs did not change significantly in the cell cycle. In the G1, S, and G2/M phases, MAPKs were phosphorylated on both tyrosine and threonine residues and dephosphorylated in metaphase-arrested cells. Our data suggest that MAPKs may play some role in the cell cycle other than G0/G1 transition.     

Role of conserved threonine and tyrosine residues in acetylcholine binding and muscarinic receptor activation. A study with m3 muscarinic receptor point mutants.

Structure-function relationship studies of the m3 muscarinic acetylcholine receptor have recently identified a series of threonine and tyrosine residues (all located within the hydrophobic receptor core) that are critically involved in acetylcholine binding (Wess, J., Gdula, D., and Brann, M.R. (1991) EMBO J. 10, 3729-3734). To gain further insight into the functional roles of these amino acids, the agonist binding properties of six rat m3 muscarinic receptor point mutants, in which the critical threonine and tyrosine residues had been individually replaced by alanine and phenylalanine, respectively, were studied in greater detail following their transient expression in COS-7 cells. The binding profiles of a series of acetylcholine derivatives suggest that the altered threonine and tyrosine residues are primarily involved in the interaction of the acetylcholine ester moiety with the receptor protein. The two m3 receptor point mutants, Thr234----Ala and Tyr506----Phe, which showed the most pronounced decreases in acetylcholine binding affinities (approximately 40-60-fold as compared with the wild-type receptor), were stably expressed in CHO cells for further functional analysis. Both mutant receptors were found to be severely impaired in their ability to stimulate agonist-dependent phosphatidylinositol hydrolysis. Consistent with this observation, acetylcholine binding to the two mutant receptors was not significantly affected by addition of the GTP analog Gpp(NH)p (5'-guanylyl imidodiphosphate). Our data suggest that Thr234 and Tyr506 (located within transmembrane domains V and VI, respectively), which are conserved among all muscarinic receptors (m1-m5), may play an important role in agonist-induced muscarinic receptor activation.     

Multiple implications of 3-phosphoinositide-dependent protein kinase 1 in human cancer

3-phosphoinositide-dependent protein kinase-1 (PDK1) is a central mediator of cellular signaling between phosphoinositide-3 kinase and various intracellular serine/threonine kinases, including protein kinase B, p70 ribosomal S6 kinase, serum and glucocorticoid-inducible kinase, and protein kinase C. PDK1 activates members of the AGC family of protein kinases by phosphorylating serine/threonine residues in the activation loop. Here, we review the regulatory mechanisms of PDK1 and its roles in cancer. PDK1 is activated by autophosphorylation in the activation loop and other serine residues, as well as by phosphorylation of Tyr-9 and Tyr-373/376. Src appears to recognize PDK1 following tyrosine phosphorylation. The role of heat shock protein 90 in regulating PDK1 stability and PDK1-Src complex formation are also discussed. Furthermore, we summarize the subcellular distribution of PDK1. Finally, an important role for PDK1 in cancer chemotherapy is proposed. In conclusion, a better understanding of its molecular regulatory mechanisms in various signaling pathways will help to explain how PDK1 acts as an oncogenic kinase in various cancers, and will contribute to the development of novel cancer chemotherapies.     

Substrate-trapping techniques in the identification of cellular PTP targets

Tyrosine phosphorylation is negatively regulated by the protein-tyrosine phosphatases (PTPs). In order to find the physiological substrates of these enzymes, diverse PTP mutants that do not possess any catalytic activities but appear to bind tightly to their tyrosine phosphorylated substrates have been designed. Hence, they can be used as tools to pull out their respective substrates from heterogeneous extracts. Named PTP "substrate-trapping" mutants by the Tonks laboratory, they represent a diverse variety of defective PTPs that are epitomized by the Cys to Ser mutant (C/S) where the active cysteine residue of the signature motif is mutated to a serine residue. In addition, new mutants have been developed which are expected to help characterize novel and less abundant substrates. In this article, we review and describe all the different substrate-trapping mutants that have successfully been used or that hold interesting promises. We present their methodology to identify substrates in vivo (co-immunoprecipitation) and in vitro (GST pulldown), and provide a current list of substrates that have been identified using these technologies.     

An investigation of cysteine mutants on the cytoplasmic loop X/XI in the melibiose transporter of Escherichia coli by using thiol reagents: implication of structural conservation of charged residues

The melibiose transporter (Mel B) of Escherichia coli is a cation-coupled (H(+), Li(+), and Na(+)) membrane protein (MW 50 kDa) consisting of 12 transmembrane helices that are connected by periplasmic and cytoplasmic loops, with both the C- and N-ends located on the cytoplasmic side of the membrane. Previous investigations on the largest cytoplasmic loop X/XI indicated that it is a functional re-entrant loop. In this communication, the cysteine mutants on loop X/XI were studied with charged thiol reagents MTSES, MTSET, and IAA for both the inhibition patterns and charge replacement/function rescue of inactive mutants in which the original charged residues were replaced by neutral cysteines. Strong inhibitions were observed in T373C and V376C by both MTSES and MTSET, consistent with previous results of PCMBS inhibition. The thiol reagents failed to recover the activities of inactive mutants D351C, D354C, and R363C and to inhibit active mutants E357C, K359C, and E365C to any significant extent, suggesting a structural conservation at D351, D354, and R363 and tolerance of structural variations at E357, K359, and E365. The results are consistent with previous observation of structural conservation of functionally charged residues in the transmembrane domains and extend to a loop the contention that in the melibiose transporter functionally important charged residues are structurally conserved.     

Refolding of human beta-1-2 GlcNAc transferase (GnT1) and the role of its unpaired Cys 121

Human beta1-2N-acetylglucosaminyltransferase (hGnT1) lacking the first 103 amino acids was expressed as a maltose binding protein (MBP) fusion protein in inclusion bodies (IBs) in Escherichia coli and refolded using an oxido-shuffling method. GnT1 mutants were prepared by replacing a predicted unpaired cysteine (C121) with alanine (C121A), serine (C121S), threonine (C121T) or aspartic acid (C121D). A double mutant R120A/C121H, was generated to mimic Gly14, the Caenorhabditis elegans GnT1 counterpart to hGNT1. Each mutant hGnT1 was constructed as an MBP fusion protein and resultant IBs were isolated and refolded. Wild type hGnT1 and mutants C121A, C121S and R120A/C121H transferred UDP-GlcNAc to the glycoprotein acceptor Man(5)-RNAse B, whereas mutants C121T and C121D were inactive. These findings indicated that cysteine 121 has a structural role in maintaining active site geometry of hGnT1, rather than a catalytic role, and illustrates for the first time the potential utility of E. coli as an expression system for hGnT1.     

The conserved active site proline determines the reducing power of Staphylococcus aureus thioredoxin

Nature uses thioredoxin-like folds in several disulfide bond oxidoreductases. Each of them has a typical active site Cys-X-X-Cys sequence motif, the hallmark of thioredoxin being Trp-Cys-Gly-Pro-Cys. The intriguing role of the highly conserved proline in the ubiquitous reducing agent thioredoxin was studied by site-specific mutagenesis of Staphylococcus aureus thioredoxin (Sa_Trx). We present X-ray structures, redox potential, pK(a), steady-state kinetic parameters, and thermodynamic stabilities. By replacing the central proline to a threonine/serine, no extra hydrogen bonds with the sulphur of the nucleophilic cysteine are introduced. The only structural difference is that the immediate chemical surrounding of the nucleophilic cysteine becomes more hydrophilic. The pK(a) value of the nucleophilic cysteine decreases with approximately one pH unit and its redox potential increases with 30 mV. Thioredoxin becomes more oxidizing and the efficiency to catalyse substrate reduction (k(cat)/K(M)) decreases sevenfold relative to wild-type Sa_Trx. The oxidized form of wild-type Sa_Trx is far more stable than the reduced form over the whole temperature range. The driving force to reduce substrate proteins is the relative stability of the oxidized versus the reduced form Delta(T(1/2))(ox/red). This driving force is decreased in the Sa_Trx P31T mutant. Delta(T(1/2))(ox/red) drops from 15.5 degrees C (wild-type) to 5.8 degrees C (P31T mutant). In conclusion, the active site proline in thioredoxin determines the driving potential for substrate reduction.     

Serine phosphorylation regulates disabled-1 early isoform turnover independently of Reelin

The Reelin-Disabled 1 (Dab1) signaling pathway plays an important role in neuronal cell migration during brain development. Dab1, an intracellular adapter protein which is tyrosine phosphorylated upon Reelin stimulation, has been directly implicated in the transmission and termination of Reelin-mediated signaling. Two main forms of Dab1 have been identified in the developing chick retina, an early isoform (Dab1-E) expressed in progenitor cells and a late isoform (Dab1-L, a.k.a. Dab1) expressed in differentiated cells. Dab1-E is missing two Src family kinase (SFK) phosphorylation sites that are critical for Reelin-Dab1 signaling and is not tyrosine phosphorylated. We have recently demonstrated a role for Dab1-E in the maintenance of retinal progenitor cells. Here, we report that Dab1-E is phosphorylated at serine/threonine residues independent of Reelin. Cdk2, highly expressed in retinal progenitor cells, mediates Dab1-E phosphorylation at serine 475 which in turn promotes ubiquitination-triggered proteasome degradation of Dab1-E. Inhibition of protein phosphatase 1 and/or protein phosphatase 2A leads to increased Dab1-E instability. We propose that Dab1 turnover is regulated by both Reelin-independent serine/threonine phosphorylation and Reelin-dependent tyrosine phosphorylation.     

Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques.

We have produced several mutants of Escherichia coli thioredoxin (Trx) using a combined mutagenesis/chemical modification technique. The protein C32S, C35S, L78C Trx was produced using standard mutagenesis procedures. After unfolding the protein with guanidine hydrochloride (GdmCl), the normally buried cysteine residue was modified with a series of straight chain aliphatic thiosulfonates, which produced cysteine disulfides to methane, ethane, 1-n-propane, 1-n-butane, and 1-n-pentane thiols. These mutants all show native-like CD spectra and the ability to activate T7 gene 5 protein DNA polymerase activity. In addition, all mutants show normal unfolding transitions in GdmCl solutions. However, the midpoint of the transition, [GdmCl]1/2, and the free energy of unfolding at zero denaturant concentration, delta G(H2O), give inverse orders of stability. This effect is due to changes in m, the dependence of delta G0 unfolding on the GdmCl concentration. The method described here may be used to produce unnatural amino acids in the hydrophobic cores of proteins.