Conserved Threonine Residue Research Articles

Radixin: Roles in the Nervous System and Beyond.

Radixin is an ERM family protein that includes radixin, moesin, and ezrin. The importance of ERM family proteins has been attracting more attention, and studies on the roles of ERM in biological function and the pathogenesis of some diseases are accumulating. In particular, we have found that radixin is the most dramatically changed ERM protein in elevated glucose-treated Schwann cells. We systemically review the literature on ERM, radixin in focus, and update the roles of radixin in regulating cell morphology, interaction, and cell signaling pathways. The potential of radixin as a therapeutic target in neurodegenerative diseases and cancer was also discussed. Radixin research has focused on its cell functions, activation, and pathogenic roles in some diseases. Radixin and other ERM proteins maintain cell shape, growth, and motility. In the nervous system, radixin has been shown to prevent neurodegeneration and axonal growth. The activation of radixin is through phosphorylation of its conserved threonine residues. Radixin functions in cell signaling pathways by binding to membrane proteins and relaying the cell signals into the cells. Deficiency of radixin has been involved in the pathogenic process of diseases in the central nervous system and diabetic peripheral nerve injury. Moreover, radixin also plays a role in cell growth and drug resistance in multiple cancers. The trials of therapeutic potential through radixin modulation have been accumulating. However, the exact mechanisms underlying the roles of radixin are far from clarification. Radixin plays various roles in cells and is involved in developing neurodegenerative diseases and many types of cancers. Therefore, radixin may be considered a potential target for developing therapeutic strategies for its related diseases. Further elucidation of the function and the cell signaling pathways that are linked to radixin may open the avenue to finding novel therapeutic strategies for diseases in the nervous system and other body systems.

Conserved linear motif within the immediate early protein ORF45 promotes its engagement with KSHV lytic cycle-promoting forkhead transcription factors, FOXK1 and FOXK2.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that can cause several cancers, such as Kaposi sarcoma and primary effusion lymphoma (PEL). We and others have recently demonstrated that Forkhead box (FOX) transcription factors can be dysregulated by KSHV, and they can affect KSHV infection. Herein, we focus on dissecting the role of two FOXK subfamily members, FOXK1 and FOXK2, in the KSHV life cycle. FOXK proteins are key host regulators of cellular functions, yet their role in KSHV infection remains unknown. Here, we demonstrated that both FOXK proteins are essential for efficient KSHV lytic reactivation in PEL cells. FOXK1 and FOXK2 are unique as they are the only FOX proteins that contain a Forkhead-associated (FHA) domain. The FHA domain is a specialized protein binding domain that recognizes a short linear serine/threonine-rich (S/T) motif. Through an unbiased motif survey, we found that KSHV viral protein ORF45 and its gammaherpesvirus homologs contain a putative FHA-binding motif. ORF45 is an immediate early tegument protein, vital for lytic reactivation and virus production. We demonstrated that ORF45 uses its novel conserved motif to interact with the FHA domain containing FOXK factors in the nucleus of infected cells. A single-point mutation of the conserved threonine residue in the motif within ORF45 abolished the ORF45-FOXK1/2 interaction. Our data indicates that FOXK proteins interact with ORF45 homologs encoded by murine gammaherpesvirus 68 (MHV68) and Rhesus macaque rhadinovirus (RRV), and that the FHA domains of FOXK proteins are sufficient for their interactions, highlighting a conserved mechanism.IMPORTANCEThe dysregulation of Forkhead transcription factors contributes to many different human diseases, including cancers, but their impact on herpesvirus lifecycle and pathogenesis is less understood. Our study uncovers a critical pro-lytic function of the FOXK subfamily and its requirement for KSHV lytic reactivation in PEL. We found that FOXK proteins bind to a key immediate early KSHV protein ORF45 using its novel short linear S/T motif. Notably, even though ORF45 homologs in gammaherpesviruses are highly diverse, we identified a similar S/T short linear motif in ORF45 homologs and also showed an evolutionary conserved interaction between FOXK proteins and ORF45 homologs of MHV68 and RRV. Our study provides a basis for future studies in animal models to evaluate the role of FOXK proteins and the impact of their interactions with ORF45 in gammaherpesvirus infection and pathogenesis. Targeting these interactions could allow a novel way to limit gammaherpesvirus infections.

Discovery, characterization, and comparative analysis of new UGT72 and UGT84 family glycosyltransferases

Glycosylated derivatives of natural product polyphenols display a spectrum of biological activities, rendering them critical for both nutritional and pharmacological applications. Their enzymatic synthesis by glycosyltransferases is frequently constrained by the limited repertoire of characterized enzyme-catalyzed transformations. Here, we explore the glycosylation capabilities and substrate preferences of newly identified plant uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) within the UGT72 and UGT84 families, with particular focus on natural polyphenol glycosylation from UDP-glucose. Four UGTs are classified according to their phylogenetic relationships and reaction products, identifying them as biocatalysts for either glucoside (UGT72 enzymes) or glucose ester (UGT84 members) formation from selected phenylpropanoid compounds. Detailed kinetic evaluations expose the unique attributes of these enzymes, including their specific activities and regio-selectivities towards diverse polyphenolic substrates, with product characterizations validating the capacity of UGT84 family members to perform di-O-glycosylation on flavones. Sequence analysis coupled with structural predictions through AlphaFold reveal an unexpected absence of a conserved threonine residue across all four enzymes, a trait previously linked to pentosyltransferases. This comparative analysis broadens the understood substrate specificity range for UGT72 and UGT84 enzymes, enhancing our understanding of their utility in the production of natural phenolic glycosides. The findings from this in-depth characterization provide valuable insights into the functional versatility of UGT-mediated reactions.

Leader peptide removal in lasso peptide biosynthesis based on penultimate isoleucine residue.

Lasso peptides are ribosomally synthesized peptides that undergo post-translational modifications including leader peptide removal by B (or the segregated B1 and B2) proteins and core peptide macrolactamization by C proteins to form a unique lariat topology. A conserved threonine residue at the penultimate position of leader peptide is hitherto found in lasso peptide precursors and shown to be a critical recognition element for effective enzymatic processing. We identified a lasso peptide biosynthetic gene cluster (bsf) from Bradymonas sediminis FA350, a Gram-negative and facultatively prey-dependent bacterium that belongs to a novel bacterial order Bradymonadales in the class Deltaproteobacteria. The kinase BsfK specifically catalyzes the phosphorylation of the precursor peptide BsfA on the Ser3 residue. BsfB1 performs dual functions to accelerate the post-translational phosphorylation and assist BsfB2 in leader peptide removal. Most importantly, the penultimate residue of leader peptide is an isoleucine rather than the conserved threonine and this isoleucine has a marked impact on the phosphorylation of Ser3 as well as leader peptide removal, implying that BsfB1 and BsfB2 exhibit a new substrate selectivity for leader peptide binding and excision. This is the first experimentally validated penultimate isoleucine residue in a lasso peptide precursor to our knowledge. In silico analysis reveals that the leader peptide Ile/Val(-2) residue is rare but not uncommon in phosphorylated lasso peptides, as this residue is also discovered in Acidobacteriaceae and Sphingomonadales in addition to Bradymonadales.

Interaction between cucumber green mottle mosaic virus MP and CP promotes virus systemic infection.

The movement protein (MP) and coat protein (CP) of tobamoviruses play critical roles in viral cell-to-cell and long-distance movement, respectively. Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus. The functions of CGMMV MP and CP during viral infection remain largely unclear. Here, we show that CGMMV MP can interact with CP in vivo, and the amino acids at positions 79-128 in MP are vital for the MP-CP interaction. To confirm this finding, we mutated five conserved residues within the residue 79-128 region and six other conserved residues flanking this region, followed by in vivo interaction assays. The results showed that the conserved threonine residue at the position 107 in MP (MPT107 ) is important for the MP-CP interaction. Substitution of T107 with alanine (MPT107A ) delayed CGMMV systemic infection in Nicotiana benthamiana plants, but increased CGMMV local accumulation. Substitutions of another 10 conserved residues, not responsible for the MP-CP interaction, with alanine inhibited or abolished CGMMV systemic infection, suggesting that these 10 conserved residues are possibly required for the MP movement function through a CP-independent manner. Moreover, two movement function-associated point mutants (MPF17A and MPD97A ) failed to cause systemic infection in plants without impacting on the MP-CP interaction. Furthermore, we have found that co-expression of CGMMV MP and CP increased CP accumulation independent of the interaction. MP and CP interaction inhibits the salicylic acid-associated defence response at an early infection stage. Taken together, we propose that the suppression of host antiviral defence through the MP-CP interaction facilitates virus systemic infection.

Cdk8 attenuates lipogenesis by inhibiting SREBP-dependent transcription in Drosophila.

Fine-tuning of lipogenic gene expression is important for the maintenance of long-term homeostasis of intracellular lipids. The SREBP family of transcription factors are master regulators that control the transcription of lipogenic and cholesterogenic genes, but the mechanisms modulating SREBP-dependent transcription are still not fully understood. We previously reported that CDK8, a subunit of the transcription co-factor Mediator complex, phosphorylates SREBP at a conserved threonine residue. Here, using Drosophila as a model system, we observed that the phosphodeficient SREBP proteins (SREBP-Thr390Ala) were more stable and more potent in stimulating the expression of lipogenic genes and promoting lipogenesis in vivo than wild-type SREBP. In addition, starvation blocked the effects of wild-type SREBP-induced lipogenic gene transcription, whereas phosphodeficient SREBP was resistant to this effect. Furthermore, our biochemical analyses identified six highly conserved amino acid residues in the N-terminus disordered region of SREBP that are required for its interactions with both Cdk8 and the MED15 subunit of the small Mediator complex. These results support that the concerted actions of Cdk8 and MED15 are essential for the tight regulation of SREBP-dependent transcription. This article has an associated First Person interview with the first author of the paper.

Maize CDKA2;1a and CDKB1;1 kinases have different requirements for their activation and participate in substrate recognition.

Cyclin-dependent kinases (CDKs), in association with cyclins, control cell cycle progression by phosphorylating a large number of substrates. In animals, activation of CDKs regularly requires both the association with a cyclin and then phosphorylation of a highly conserved threonine residue in the CDK activation loop (the classical mechanism), mediated by a CDK-activating kinase (CAK). In addition to this typical mechanism of activation, some CDKs can also be activated by the association of a cyclin to a monomeric CDK previously phosphorylated by CAK although not all CDKs can be activated by this mechanism. In animals and yeast, cyclin, in addition to being required for CDK activation, provides substrate specificity to the cyclin/CDK complex; however, in plants both the mechanisms of CDKs activation and the relevance of the CDK-associated cyclin forsubstrate targeting have been poorly studied. In this work, by co-expressing proteins in E.coli, we studied maize CDKA2;1a and CDKB1;1, two of the main types of CDKs that control the cell cycle in plants. These kinases could be activated by the classical mechanism and by the association of CycD2;2a to a phosphorylated intermediate in its activation loop, a previously unproven mechanism for the activation of plant CDKs. Unlike CDKA2;1a, CDKB1;1 did not require CAK for its activation, since it autophosphorylated in its activation loop. Phosphorylation of CDKB1;1 and association of CycD2;2 was not enough for its full activation as association of maize CKS, a scaffolding protein, differentially stimulated substrate phosphorylation. Our results suggest that both CDKs participate in substrate recognition.

LRRK2 phosphorylation of Rab GTPases in Parkinson's disease.

Rab GTPases comprise a large family of conserved GTPases that are critical regulators of the secretory and endocytic pathways. The human genome encodes ~ 65 Rabs that localize to discrete membrane compartments and, when in their GTP-bound state, bind to effector proteins to carry out diverse functions. Activating mutations in LRRK2 kinase cause Parkinson's disease, and subsets of Rab GTPases are important LRRK2 substrates. LRRK2 phosphorylates a conserved threonine residue that is essential for Rab interaction with guanine nucleotide exchange factors, effectors, and GDI that recycles Rabs between membrane compartments. This brief review will highlight new findings related to LRRK2-mediated phosphorylation of Rab GTPases and its consequences. Remarkably, Rab phosphorylation flips a switch on Rab effector selection with dominant consequences for cell pathophysiology.

Clostridiumnovyi's Alpha-Toxin Changes Proteome and Phosphoproteome of HEp-2 Cells.

C. novyi type A produces the alpha-toxin (TcnA) that belongs to the large clostridial glucosylating toxins (LCGTs) and is able to modify small GTPases by N-acetylglucosamination on conserved threonine residues. In contrast, other LCGTs including Clostridioides difficile toxin A and toxin B (TcdA; TcdB) modify small GTPases by mono-o-glucosylation. Both modifications inactivate the GTPases and cause strong effects on GTPase-dependent signal transduction pathways and the consequent reorganization of the actin cytoskeleton leading to cell rounding and finally cell death. However, the effect of TcnA on target cells is largely unexplored. Therefore, we performed a comprehensive screening approach of TcnA treated HEp-2 cells and analyzed their proteome and their phosphoproteome using LC-MS-based methods. With this data-dependent acquisition (DDA) approach, 5086 proteins and 9427 phosphosites could be identified and quantified. Of these, 35 proteins were found to be significantly altered after toxin treatment, and 1832 phosphosites were responsive to TcnA treatment. By analyzing the TcnA-induced proteomic effects of HEp-2 cells, 23 common signaling pathways were identified to be altered, including Actin Cytoskeleton Signaling, Epithelial Adherens Junction Signaling, and Signaling by Rho Family GTPases. All these pathways are also regulated after application of TcdA or TcdB of C. difficile. After TcnA treatment the regulation on phosphorylation level was much stronger compared to the proteome level, in terms of both strength of regulation and the number of regulated phosphosites. Interestingly, various signaling pathways such as Signaling by Rho Family GTPases or Integrin Signaling were activated on proteome level while being inhibited on phosphorylation level or vice versa as observed for the Role of BRCA1 in DNA Damage Response. ZIP kinase, as well as Calmodulin-dependent protein kinases IV & II, were observed as activated while Aurora-A kinase and CDK kinases tended to be inhibited in cells treated with TcnA based on their substrate regulation pattern.

STK25 functions as an IRF5 kinase to promote TLR7/8-mediated inflammation

Abstract Toll-like receptors (TLRs) represent a subset of pathogen-recognition receptors (PRRs) employed by the innate immune system to detect pathogen-associated molecular patterns (PAMPs) and initiate the response to invading microbes. The transcription factor interferon regulatory factor 5 (IRF5) functions as an important mediator of the inflammatory response downstream of myeloid differentiation factor 88 (MyD88)-dependent TLR activation. While the dysregulation of IRF5 activity has been implicated in the development of several autoimmune diseases including systemic lupus erythematosus and rheumatoid arthritis, the factors that modulate TLR-induced IRF5 post-translational modifications (PTMs) are poorly understood. Therefore, the focus of this study is to identify protein kinases involved in the regulation of TLR7/8 signaling. We performed a kinome-based siRNA screen in human THP-1 monocytic cells and human primary myeloid cells to identify important mediators of TLR7/8-induced TNF-α and IL-6 production. We identified serine/threonine protein kinase 25 (STK25) as a positive regulator of proinflammatory cytokine production in response to TLR7/8 activation in human myeloid cells. We determined that STK25 phosphorylates IRF5 in vitro via multiple biochemical assays. Phosphopeptide mapping by mass spectrometry revealed that STK25 phosphorylates IRF5 at a highly conserved threonine residue. Thus, our data implicate STK25 as a regulator of TLR7/8 signaling through the modulation of IRF5 activity.

The FocA channel functions to maintain intracellular formate homeostasis during Escherichia coli fermentation.

FocA translocates formate/formic acid bi-directionally across the cytoplasmic membrane when Escherichia coli grows by fermentation. It remains unclear, however, what physiological benefit is imparted by FocA, because formic acid (pKa=3.75) can diffuse passively across the membrane, especially at low pH. Here, we monitored changes in intra- and extracellular formate levels during batch-culture fermentation, comparing a parental E. coli K-12 strain with its isogenic focA mutant. Our results show that, regardless of the initial pH in the culture, FocA functions to maintain relatively constant intracellular formate levels during growth. Analysis of a strain synthesizing a FocAT91A variant with an exchange in a conserved threonine residue within the translocation pore revealed the strain accumulated formate intracellularly and imported formate poorly, but in a pH-dependent manner, which was different to uptake by native FocA. We conclude that FocA maintains formate homeostasis, using different mechanisms for efflux and uptake of the anion.

Structural Insight into the Reaction Mechanism of Ketosynthase-Like Decarboxylase in a Loading Module of Modular Polyketide Synthases.

Ketosynthase-like decarboxylase (KSQ) domains are widely distributed in the loading modules of modular polyketide synthases (PKSs) and are proposed to catalyze the decarboxylation of a malonyl or methylmalonyl unit for the construction of the PKS starter unit. KSQ domains have high sequence similarity to ketosynthase (KS) domains, which catalyze transacylation and decarboxylative condensation in polyketide and fatty acid biosynthesis, except that the catalytic Cys residue of KS domains is replaced by Gln in KSQ domains. Here, we present biochemical analyses of GfsA KSQ and CmiP4 KSQ, which are involved in the biosynthesis of FD-891 and cremimycin, respectively. In vitro analysis showed that these KSQ domains catalyze the decarboxylation of malonyl and methylmalonyl units. Furthermore, we determined the crystal structure of GfsA KSQ in complex with a malonyl thioester substrate analogue, which enabled identification of key amino acid residues involved in the decarboxylation reaction. The importance of these residues was confirmed by mutational analysis. On the basis of these findings, we propose a mechanism of the decarboxylation reaction catalyzed by GfsA KSQ. GfsA KSQ initiates decarboxylation by fixing the substrate in a suitable conformation for decarboxylation. The formation of enolate upon decarboxylation is assisted by two conserved threonine residues. Comparison of the structure of GfsA KSQ with those of KS domains suggests that the Gln residue in the active site of the KSQ domain mimics the acylated Cys residue in the active site of KS domains.

Autophosphorylation of the CK1 Kinase Domain Regulates Enzyme Activity and Substrate Specificity

CK1 enzymes are conserved, acidophilic serine/threonine kinases with a variety of critical cellular functions; misregulation of CK1 contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Despite this, little is known about how CK1 activity is controlled. CK1 kinases have highly similar catalytic domains, plus a conserved extension to that kinase domain that is important for enzyme stability and activity. In contrast to the catalytic domains, the C-terminal tails of CK1 family members diverge in sequence and length; however, they all appear to serve as substrates of autophosphorylation. The autophosphorylated tails are proposed to inhibit kinase activity by acting as pseudosubstrates. In addition to C-terminal autophosphorylation, autophosphorylation of the CK1Σ kinase domain has been detected, but its effect on CK1 activity and cellular function has never been explored. Here, we addressed the role of kinase domain autophosphorylation in human CK1δ and CK1Σ, as well as Hhp1 and Hhp2, their homologues in Schizosaccharomyces pombe. In each case, we found that autophosphorylation of a conserved threonine residue in the kinase domain inhibited enzyme activity. This site resides in the mobile L-EF loop proximal to the active site, distinct from the well-characterized T-loop autophosphorylation that occurs in other kinase families. We propose that phosphorylation on the L-EF loop inhibits substrate docking with the kinase domain by shielding the positively charged substrate binding pocket. Consistent with this hypothesis, yeast and human enzymes with phosphoablating mutations at this site were hyperactive in vitro. In vivo, mutation of this site protected yeast cells from heat shock, indicating a change in the phosphorylation of substrates in the stress response pathway. To determine whether other pathways may also be rewired by this mutation, we utilized quantitative phosphoproteomics to probe the substrateomes of wildtype and mutant Hhp1 and Hhp2. We found that ablating autophosphorylation in the kinase domain significantly changed the substrate profiles of these enzymes, suggesting that this regulatory mechanism influences substrate specificity in addition to catalytic activity. Due to the strong sequence conservation of this autophosphorylation site and the functional importance of the L-EF loop, which is unique to the CK1 family of kinases, this mechanism is likely to regulate the majority of CK1 enzymes in vivo. We predict that kinase domain autophosphorylation works in conjunction with other mechanisms, such as C-terminal autophosphorylation, to ultimately determine the extent to which different substrates are phosphorylated in different cellular contexts.

Pharmacologic Inhibition of Ezrin-Radixin-Moesin Phosphorylation is a Novel Therapeutic Strategy in Rhabdomyosarcoma.

Intermediate and high-risk rhabdomyosarcoma (RMS) patients have poor prognosis with available treatment options, highlighting a clear unmet need for identification of novel therapeutic strategies. Ezrin-radixin-moesin (ERM) family members are membrane-cytoskeleton linker proteins with well-defined roles in tumor metastasis, growth, and survival. ERM protein activity is regulated by dynamic changes in the phosphorylation at a conserved threonine residue in their C-terminal actin-binding domain. Interestingly, ERM family member, ezrin, has elevated expression in the RMS tissue. Despite this, the translational scope of targeting ERM family proteins in these tumors through pharmacological inhibition has never been considered. This study investigates the inhibition of ERM phosphorylation using a small molecule pharmacophore NSC668394 as a potential strategy against RMS. Upon in vitro treatment with NSC668394, RMS cells exhibit a dose-dependent decrease in cell viability and proliferation, with induction of caspase-3 cleavage and apoptosis. siRNA-mediated knockdown of individual ERM protein expression revealed that each regulates RMS survival to a different degree. In vivo administration of NSC668394 in RMS xenografts causes significant decrease in tumor growth, with no adverse effect on body weight. Collectively, this study highlights the importance of the active conformation of ERM proteins in RMS progression and survival and supports pharmacologic inhibition of these proteins as a novel therapeutic approach.

Transcriptional regulation by σ factor phosphorylation in bacteria.

A major form of transcriptional regulation in bacteria occurs through the exchange of the primary σ factor of RNA polymerase (RNAP) with an alternative extracytoplasmic function (ECF) σ factor1. ECF σ factors are generally intrinsically active and are retained in an inactive state via the sequestration into σ factor-anti-σ factor complexes until their action is warranted2-20. Here, we report a previously uncharacterized mechanism of transcriptional regulation that relies on intrinsically inactive ECF σ factors, the activation of which and interaction with the β'-subunit of RNAP depends on σ factor phosphorylation. In Vibrio parahaemolyticus, the threonine kinase PknT phosphorylates the σ factor EcfP, which results in EcfP activation and expression of an essential polymyxin-resistant regulon. EcfP phosphorylation occurs at a highly conserved threonine residue, Thr63, positioned within a divergent region in the σ2.2 helix. Our data indicate that EcfP is intrinsically inactive and unable to bind the β'-subunit of RNAP due to the absence of a negatively charged DAED motif in this region. Furthermore, our results indicate that phosphorylation at residue Thr63 mimics this negative charge and licenses EcfP to interact with the β'-subunit in the formation of the RNAP holoenzyme, which in turn results in target gene expression. This regulatory mechanism is a previously unrecognized paradigm in bacterial signal transduction and transcriptional regulation, and our data suggest that it is widespread in bacteria.

PUB25 and PUB26 Promote Plant Freezing Tolerance by Degrading the Cold Signaling Negative Regulator MYB15.

Cold stress adversely affects plant growth, development, and crop productivity and quality. Plants employ cold acclimation strategy to protect them from cold damage. The transcription-factor-CBF-dependent cold signaling pathway plays a key role in plant cold acclimation. However, how this signaling pathway is dynamically and precisely regulated remains unknown. Here, we report that two U-box type E3 ubiquitin ligases, PUB25 and PUB26, positively regulate freezing tolerance in Arabidopsis thaliana. Both PUB25 and PUB26 poly-ubiquitinate MYB15, a transcriptional repressor of the CBF-dependent cold signaling pathway, leading to MYB15 degradation and thus enhanced CBF expression under cold stress. Furthermore, cold-activated OST1 specifically phosphorylates PUB25 and PUB26 at conserved threonine residues, enhancing their E3 activity and facilitating the cold-induced degradation of MYB15. Our results thus unravel the regulatory role of the OST1-PUB25/26 module in regulating the duration and amplitude of the cold response by controlling the homeostasis of the negative regulator MYB15.

Protein kinase A inhibits tumor mutator APOBEC3B through phosphorylation

APOBEC3B cytidine deaminase (A3B) catalyzes cytosine into uracil in single-strand DNA and induces C-to-T mutations in genomic DNA of various types of tumors. Accumulation of APOBEC signature mutations is correlated with a worse prognosis for patients with breast cancer or multiple myeloma, suggesting that A3B activity might be a cause of the unfavorable DNA mutations and clonal evolution in these tumors. Phosphorylation of conserved threonine residues of other cytidine deaminases, activation induced deaminase (AID) and APOBEC3G, inhibits their activity. Here we show that protein kinase A (PKA) physically binds to A3B and phosphorylates Thr214. In vitro deaminase assays and foreign DNA editing assays in cells confirm that phosphomimetic A3B mutants, T214D and T214E, completely lose deaminase activity. Molecular dynamics simulation of A3B phosphorylation reveals that Thr214 phosphorylation disrupts binding between the phospho-A3B catalytic core and ssDNA. These mutants still inhibit retroviral infectivity at least partially, and also retain full anti-retrotransposition activity. These results imply that PKA-mediated phosphorylation inhibits A3B mutagenic activity without destructing its innate immune functions. Therefore, PKA activation could reduce further accumulation of mutations in A3B overexpressing tumors.

Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration

BackgroundMutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson’s disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the substrates that mediate neurodegeneration remain poorly defined. Given the increasing evidence suggesting a role of LRRK2 in membrane and vesicle trafficking, here we systemically screened Rab GTPases, core regulators of vesicular dynamics, as potential substrates of LRRK2 and investigated the functional consequence of such phosphorylation in cells and in vivo.MethodsIn vitro LRRK2 kinase assay with forty-five purified human Rab GTPases was performed to identify Rab family proteins as substrates of LRRK2. We identified the phosphorylation site by tandem mass-spectrometry and confirmed it by assessing phosphorylation in the in vitro LRRK2 kinase assay and in cells. Effects of Rab phosphorylation on neurodegeneration were examined in primary cultures and in vivo by intracranial injection of adeno-associated viral vectors (AAV) expressing wild-type or phosphomutants of Rab35.ResultsOur screening revealed that LRRK2 phosphorylated several Rab GTPases at a conserved threonine residue in the switch II region, and by using the kinase-inactive LRRK2-D1994A and the pathogenic LRRK2-G2019S along with Rab proteins in which the LRRK2 site was mutated, we verified that a subset of Rab proteins, including Rab35, were authentic substrates of LRRK2 both in vitro and in cells. We also showed that phosphorylation of Rab regulated GDP/GTP-binding property in cells. Moreover, in primary cortical neurons, mutation of the LRRK2 site in several Rabs caused neurotoxicity, which was most severely induced by phosphomutants of Rab35. Furthermore, intracranial injection of the AAV-Rab35 -T72A or AAV-Rab35-T72D into the substantia nigra substantially induced degeneration of dopaminergic neurons in vivo.ConclusionsHere we show that a subset of Rab GTPases are authentic substrates of LRRK2 both in vitro and in cells. We also provide evidence that dysregulation of Rab phosphorylation in the LRRK2 site induces neurotoxicity in primary neurons and degeneration of dopaminergic neurons in vivo. Our study suggests that Rab GTPases might mediate LRRK2 toxicity in the progression of PD.

Akt regulates neurite growth by phosphorylation-dependent inhibition of radixin proteasomal degradation

Neurite growth is controlled by a complex molecular signaling network that regulates filamentous actin (F-actin) dynamics at the growth cone. The evolutionarily conserved ezrin, radixin, and moesin family of proteins tether F-actin to the cell membrane when phosphorylated at a conserved threonine residue and modulate neurite outgrowth. Here we show that Akt binds to and phosphorylates a threonine 573 residue on radixin. Akt-mediated phosphorylation protects radixin from ubiquitin-dependent proteasomal degradation, thereby enhancing radixin protein stability, which permits proper neurite outgrowth and growth cone formation. Conversely, the inhibition of Akt kinase or disruption of Akt-dependent phosphorylation reduces the binding affinity of radixin to F-actin as well as lowers radixin protein levels, resulting in decreased neurite outgrowth and growth cone formation. Our findings suggest that Akt signaling regulates neurite outgrowth by stabilizing radixin interactions with F-actin, thus facilitating local F-actin dynamics.

Erythropoietin Signaling Regulates Heme Biosynthesis

Heme plays a fundamental role in a diverse array of cellular processes and is required for the survival of all cells. During erythropoiesis, heme production is drastically upregulated to support the production of oxygen-carrying hemoglobin. This increase in heme production is mediated by transcriptional induction of heme metabolism genes including ferrochelatase (FECH), which is the enzyme that catalyzes the rate-limiting insertion of ferrous iron into protoporphyrin IX in the mitochondria of erythroid cells. However, how heme production is coordinately regulated by extracellular cues is currently unknown.Here, using complementary biochemical and genetics approaches, we show that erythropoietin (EPO) signaling regulates heme biosynthesis via a protein kinase A (PKA)-dependent mechanism. In its inactive state, PKA is a tetrameric complex consisting of two catalytic subunits (C) that are bound to and inhibited by two regulatory subunits (R). The C subunits become activated to phosphorylate downstream target proteins when they dissociate from the R subunits. We demonstrate that EPO-induced JAK2 (janus kinase 2) activity leads to release of the C subunits from the R subunits. We also find that phosphorylated STAT5 (signal transducer and activator of transcription 5) forms a molecular complex with PKA-C. This suggests that phospho-STAT5 can outcompete PKA-R to release PKA-C to directly phosphorylate FECH at a highly conserved threonine residue located in the catalytic site. We examined the importance of FECH phosphorylation in vivo by taking advantage of CRISPR/Cas9-mediated genome editing to knock-in the analogous Thr115Ala substitution into the endogenous Fech gene in murine RBCs. Erythroid cells harboring the homozygous Thr115Ala Fech mutation exhibited a block in hemoglobin production and concomitant intracellular accumulation of upstream protoporphyrin intermediates. Strikingly, this phenotype bears resemblance to erythropoietic protoporphyria (EPP), a human hematologic disorder typically associated with FECHmutations.Together, our results support a model where EPO signaling during erythroid maturation activates PKA by a previously unrecognized JAK2/STAT5-dependent mechanism. Phosphorylation of FECH is required for full activity to support elevated heme biosynthesis and hemoglobin production. Furthermore, our data implicates aberrant EPO/JAK2/PKA signaling in the pathogenesis of human EPP. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.