CyclinA2-Cyclin-dependent Kinase Regulates SAMHD1 Protein Phosphohydrolase Domain

Yan, Junpeng; Hao, Caili; DeLucia, Maria; Swanson, Selene K.; Florens, Laurence; Washburn, Michael P.; Ahn, Jin-Woo; Skowroński, Jacek

doi:10.1074/jbc.m115.646588

Cited by 89 publications

(137 citation statements)

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“…Previous work by Yan et al showed that pT592 is present in S and G2/M in subpopulations of leukocytes isolated by cell sorting [18]. Here we followed the phosphorylation pattern of T592 during cell cycle progression in synchronized living cells and found that it is intimately linked to kinase and phosphatase activities controlling cell cycle progression.…”

Section: Discussionmentioning

confidence: 59%

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The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

et al. 2018

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SAMHD1 is the major catabolic enzyme regulating the intracellular concentrations of DNA precursors (dNTPs). The S-phase kinase CDK2-cyclinA phosphorylates SAMHD1 at Thr-592. How this modification affects SAMHD1 function is highly debated. We investigated the role of endogenous SAMHD1 phosphorylation during the cell cycle. Thr-592 phosphorylation occurs first at the G1/S border and is removed during mitotic exit parallel with Thr-phosphorylations of most CDK1 targets. Differential sensitivity to the phosphatase inhibitor okadaic acid suggested different involvement of the PP1 and PP2 families dependent upon the time of the cell cycle. SAMHD1 turn-over indicates that Thr-592 phosphorylation does not cause rapid protein degradation. Furthermore, SAMHD1 influenced the size of the four dNTP pools independently of its phosphorylation. Our findings reveal that SAMHD1 is active during the entire cell cycle and performs an important regulatory role during S-phase by contributing with ribonucleotide reductase to maintain dNTP pool balance for proper DNA replication.

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Section: Discussionmentioning

confidence: 59%

“…In proliferating cells, none of the tested SAMHD1 variants blocked retroviral infection, presumably due to the high expression of RNR that opposed the catabolic activity of SAMHD1[22]. Interestingly, only the non-phosphorylatable SAMHD1 mutants reduced the percentage of cells in S-phase and activated the DNA damage check-point[18]. …”

Section: Introductionmentioning

confidence: 99%

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

et al. 2018

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“…The expression of phosphorylation site-mutated SAMHD1 caused cell cycle delay and DNA damage (33). ISF2 is similarly unregulated and thus might be expected to have negative consequences.…”

Section: Discussionmentioning

confidence: 99%

“…SAMHD1 phosphorylation by CDK2 is thought to be a means of controlling dNTP levels through the cell cycle (33,34), allowing the dNTP concentration to increase during S and decrease in G 1 /G 0 . The expression of phosphorylation site-mutated SAMHD1 caused cell cycle delay and DNA damage (33).…”

Section: Discussionmentioning

confidence: 99%

A Highly Active Isoform of Lentivirus Restriction Factor SAMHD1 in Mouse

Bloch

Gläsker

Sitaram

et al. 2017

Journal of Biological Chemistry

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Edited by Henrik G. DohlmanThe triphosphohydrolase SAMHD1 (sterile ␣ motif and histidine-aspartate domain-containing protein 1) restricts HIV-1 replication in nondividing myeloid cells by depleting the dNTP pool, preventing reverse transcription. SAMHD1 is also reported to have ribonuclease activity that degrades the virus genomic RNA. Human SAMHD1 is regulated by phosphorylation of its carboxyl terminus at Thr-592, which abrogates its antiviral function yet has only a small effect on its phosphohydrolase activity. In the mouse, SAMHD1 is expressed as two isoforms (ISF1 and ISF2) that differ at the carboxyl terminus due to alternative splicing of the last coding exon. In this study we characterized the biochemical and antiviral properties of the two mouse isoforms of SAMHD1. Both are antiviral in nondividing cells. Mass spectrometry analysis showed that SAMHD1 is phosphorylated at several amino acid residues, one of which (Thr-634) is homologous to Thr-592. Phosphomimetic mutation at Thr-634 of ISF1 ablates its antiviral activity yet has little effect on phosphohydrolase activity in vitro. dGTP caused ISF1 to tetramerize, activating its catalytic activity. In contrast, ISF2, which lacks the phosphorylation site, was significantly more active, tetramerized, and was active without added dGTP. Neither isoform nor human SAMHD1 had detectable RNase activity in vitro or affected HIV-1 genomic RNA stability in newly infected cells. These data support a model in which SAMHD1 catalytic activity is regulated through tetramer stabilization by the carboxyl-terminal tail, phosphorylation destabilizing the complexes and inactivating the enzyme. ISF2 may serve to reduce the dNTP pool to very low levels as a means of restricting virus replication. The lentiviral restriction factor SAMHD12 is a dNTP triphosphohydrolase (1, 2) that inhibits the replication of HIV-1 in myeloid cells (3, 4). The enzyme removes the triphosphate groups of dNTPs, preventing the reverse transcription in newly infected cells (5). In addition, SAMHD1 has been proposed to have ribonuclease activity that degrades the viral genomic RNA upon virus entry (6). HIV-2 and some SIVs counteract the restriction by encoding Vpx, a virion-packaged accessory protein that induces the proteasomal degradation of SAMHD1 (3, 4, 7). Other SIVs, such as the SIV of African green monkeys, counteract SAMHD1 through the related accessory protein Vpr (8, 9). Vpx delivery into the cell by virions causes a rapid rise in the intracellular dNTP concentration, relieving the block to reverse transcription and allowing productive infection (5, 10). HIV-1 does not encode Vpx and as a result replicates poorly in dendritic and other myeloid cells. The requirement for Vpx for infection of myeloid cells can be partially replaced in culture by the addition of extracellular deoxynucleosides, which are converted into dNTPs by the salvage pathway (5). In addition, HIV-1 can be genetically modified to package Vpx, allowing it to infect myeloid cells (11,12).The catalytic activity of SAMHD1 is reg...

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“…Aliquots (10 5 cells) of the purified populations were reanalyzed by FACS to assess their purity. The cells were lysed (62,64), and cytoplasmic or chromatin extracts were analyzed by immunoblotting. Detailed experimental procedures are described in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins

Hrecka

Hao

Shun

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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103

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HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4DCAF1 E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here, we identify HLTF, a postreplication DNA repair helicase, as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4DCAF1 E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81, which Vpr also directs for degradation via CRL4DCAF1 E3. Thus, separate functions of HIV-1 Vpr usurp CRL4DCAF1 E3 to remove key enzymes in three DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery, suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host.

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CyclinA2-Cyclin-dependent Kinase Regulates SAMHD1 Protein Phosphohydrolase Domain

Cited by 89 publications

References 50 publications

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

A Highly Active Isoform of Lentivirus Restriction Factor SAMHD1 in Mouse

HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins

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