We used a double thymidine block to obtain satisfactory synchronization of normal human lung fibroblasts at the G1/S border. 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 CD96 role during S-phase by contributing with ribonucleotide reductase to maintain dNTP pool balance for proper DNA replication. nuclease activity were reported [3,4]. However, later data attributed the nuclease activity to contaminants co-purifying with SAMHD1 and the question of SAMHD1? harboring multiple functions is still debated [5]. SAMHD1 is expressed at variable levels in most human tissues, especially in immune cells. It has been intensively investigated as a host restriction factor that, in quiescent/differentiated cells, limits HIV-1 and other viral infections by lowering cellular dNTP concentrations under a threshold critical for the synthesis of viral DNA [6]. SAMHD1 gene mutations are associated with Methotrexate (Abitrexate) the Aicardi-Goutires syndrome (AGS), a severe inflammatory encephalopathy characterized by inappropriate immune activation [7]. Both in AGS individuals and transgenic models the loss of SAMHD1 results in increased cellular concentrations of dNTPs [8]. SAMHD1 mutations occur in leukemias [9] and other types of human cancer, suggesting that a surplus of dNTPs contributes to cell transformation by affecting the fidelity of DNA synthesis. SAMHD1 is a component of the enzyme network that controls dNTP levels [10]. In mammalian cells the concentrations of dNTPs are regulated with cell division cycle progression. During S-phase, the pools expand due to the induction of ribonucleotide reductase (RNR), the major anabolic enzyme providing deoxynucleotides for DNA replication. Outside S-phase, RNR activity is restricted by the ubiquitin-dependent degradation of its R2 subunit [11,12], with concomitant contraction of dNTP pools. In G1 and in quiescent cells, p53R2, the stable small subunit of RNR, provides dNTPs for DNA repair and mitochondrial DNA maintenance [13]. SAMHD1 is present during the whole cell cycle and prevents overproduction of dNTPs. Nevertheless, it is still unclear if SAMHD1 activity and protein concentration are regulated and whether SAMHD1 regulation is inversely related to that of RNR. SAMHD1 is phosphorylated at threonine 592 (T592) by the cell-cycle regulated kinases Methotrexate (Abitrexate) CDK2/1 [14C16]. Phosphorylated T592 is believed to have a regulatory function but how it relates to SAMHD1 activity and/or protein stability is still questioned. Biochemical studies with recombinant phosphomimetic (T592D/E) and non-phosphorylatable (T592A/V) SAMHD1 mutants yielded conflicting results regarding tetramer stability and enzymatic properties [15,17C21]. In live cells, the Methotrexate (Abitrexate) effects of SAMHD1 phosphorylation were investigated by ectopic over-expression of SAMHD1 mutants and the restriction of viral infection or dNTP pool decrease, both readouts of SAMHD1 activity. In PMA differentiated U937 cells, phosphomimetic SAMHD1 mutants lacked retroviral restriction although they decreased cellular dNTP concentrations as did wild type SAMHD1 and its non-phosphorylatable mutants [15,20C22]. 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]. No study so far has investigated SAMHD1 dephosphorylation nor looked for the protein phosphatases involved. With this background in mind we wished to address the timing and role of SAMHD1 Methotrexate (Abitrexate) phosphorylation during cell cycle progression. We chose the strategy of correlating endogenous SAMHD1 phosphorylation with the dNTP levels in the individual phases of the cell division cycle, comparing parental SAMHD1-proficient and SAMHD1-KO cell lines. We investigated the regulation of SAMHD1 phosphorylation by kinase and phosphatase activities in synchronized cultures. Moreover, we tested the possibility that T592 phosphorylation acts as a signal for degradation, by measuring the turn-over of the protein in cycling cells. We suggest that SAMHD1 is a long-lived protein, active in intact cells during the entire cell division cycle independently of T592.