PIMREG (PICALM interacting mitotic regulator)

	Figure 1: Genomic organization, alternative splicing, and protein isoforms of PIMREG (FAM64). Boxes represent exons, with blue parts symbolizing coding region and gray parts the 5' and 3' UTRs. There are 2 splice variants (sizes are indicated in parentheses). In splice variant 2, exon 5 is spliced out resulting in a longer protein (FAM64A isoform 2) encoded by exons 2, 3, 4 and 6. The FAM64A isoform 1 (shorter form) is encoded by exons 2, 3, 4 and 5 from splice variants 1 and the transcript with retained intron 5 (see in the text). Both FAM64A isoforms are identical from amino acids 1-228, they differ in their last 10 and 20 C-terminal residues (isoform 1 and 2, respectively). (Figure modified from Archangelo, et al. 2006)
Description	PIMREG (FAM64) is located on chromosome 17 band p13.2, 6.29 megabase pairs from the telomere of the short arm (chromosome 17 genomic contig NT_010718). The genomic locus spans 7 Kb and contains 6 exons with a non-coding first exon.
Transcription	Two alternatively spliced transcripts of 1.5 Kb are formed (NM_019013 and NM_001195228) and code for two protein isoforms of 238 and 248 amino acids in length (isoform 1 and 2, respectively). Both isoforms share the first 228 residues, encoded by exons 2-4. The two proteins differ in their C-termini. The 10 last amino acids of isoform 1 are encoded by exon 5, which is absent in the transcript coding for isoform 2. The last 20 amino acids of isoform 2 are encoded by exon 6 (Figure 1) (Archangelo, et al. 2006). An additional transcript variant with retained intron 5 (between exons 5 and 6) was described (Archangelo, et al. 2006; Coulombe-Huntington, et al. 2009). There are 4 processed pseudogenes of FAM64A at other chromosomal locations (2q33, 4p15, 4q24 and 6q15).

	Figure 2: Diagram representing the protein structure of PIMREG (FAM64). FAM64A is a phosphoprotein. All residues described to be phosphorylated in large scale proteomic studies are depicted. Source: Phosphoproteomic databases PhosphoSitePlus™ (http://www.phosphosite.org) and Phosida (http://www.phosida.com/). Destruction box motifs are depicted as orange boxes (DB1 and DB2). Aminoacids comprising the DB motifs and the unique phospho-residue characterized (S131) are shown. (Figure modified from Archangelo, et al. 2013)
Description	The FAM64A calculated molecular weight is about 27 kDa. The protein contains two putative destruction box motifs (RxxL) at amino acids 14-17 and 53-56 (DB1 and DB2, respectively), the second being a recognition motif for the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) (Zhao, et al. 2008). There are three types of posttranslational modifications identified within FAM64A isoforms, namely lysine- acetylation (K-ac), lysine-ubiquitination (K-ub) and phosphorylation (S-p and T-p). A total of 22 residues were described as phosphorylated in FAM64A in a variety of large scale proteomic studies as revealed by the phosphoproteomic databases PhosphoSitePlus™ (http://www.phosphosite.org) and Phosida (http://www.phosida.com/) (Figure 2).
Expression	In normal adult tissue FAM64A is predominantly expressed in thymus, spleen and colon and to a lesser extent in small intestines, ovary and brain (Archangelo, et al. 2006; Archangelo, et al. 2008). Moreover, FAM64A is highly expressed in leukemia, lymphoma and tumor cell lines. The protein levels vary throughout the cell cycle. In synchronized cells FAM64A accumulates in S and G2 phases of the cell cycle, peaks in the mitosis and drops drastically as cells exit from mitosis into the G1 phase (Archangelo, et al. 2008; Zhao, et al. 2008). FAM64A transcripts covariate with that of cell cycle regulation genes in tumorigenesis (Zhao, et al. 2008). Resting human peripheral blood lymphocytes (PBLs), which are not selected for growth in culture, do not express FAM64A protein. However, when these cells are induced to proliferate upon mitogen activation they do express FAM64A. Likewise, glioblastoma cells induced to proliferate, show a progressive upregulation of FAM64A. Conversely, FAM64A expression drastically decreases when highly proliferative leukemia cells cease to proliferate upon exposure to differentiation agents (Barbutti, et al. 2016).Thus, FAM64A expression positively correlates with the proliferative state of the cells (Archangelo, et al. 2008). Expression analysis of the murine Fam64a homolog revealed a strong expression throughout mouse embryogenesis, in particular at the developing central nervous system (CNS). The expression decreases gradually and proportionally as embryos develop to later stages (Archangelo, et al. 2008). In the hematopoietic compartment, Fam64A is widely expressed in different cell subpopulations (Archangelo, et al. 2008). Moreover, RNA-Seq analysis of individual hematopoietic stem cells (HSCs) to resolve heterogeneity within HSC population, revealed that Fam64a is highly expressed in the HSC population primed for proliferation (Wilson, et al. 2015).
Localisation	FAM64A is a nuclear protein enriched in the nucleoli. Expression levels in the nucleoplasm and nucleolar accumulation are different from cell to cell and dependent on the cell cycle phase (Archangelo, et al. 2008) (Figure 3). In mitotic cells FAM64A-GFP is diffusely distributed throughout cells without specific association with chromosomes, kinetochores, spindle, or centrosome (Zhao, et al. 2008).
	Figure 3: Subcellular localization of endogenous PIMREG (FAM64). Endogenous protein was visualized by immunofluorescence and confocal microscopy in non-synchronized U2OS cells. FAM64A is a nuclear protein enriched in the nucleolus of some cells. Note the different levels of FAM64A expression from cell to cell. FAM64A protein levels and nucleolar localization are cell cycle dependent. The figure shows cells labeled with Cy3 (FAM64A) and DAPI with channels merged.
Function	Based on the fact that levels of FAM64A protein strongly correlate with cellular proliferation in both normal and malignant cells, this protein was described as a marker for proliferation with a possible role in the control of cell proliferation and tumorigenesis (Archangelo, et al. 2008). In fact, silencing of FAM64A protein in the U937 leukemia cell line resulted in reduced proliferation and altered cell cycle progression, as attested by diminished expression of the cell cycle regulators CCNA1, CCNE1, CCNB1 (cyclin-A, -E and -B1) in FAM64A depleted cells (Barbutti, et al. 2016). Furthermore, Zhao and coworkers described FAM64A as a regulator of chromosome segregation. FAM64A is a substrate of the APC/C complex that controls the metaphase to anaphase transition and its knock-down resulted in accelerated anaphase onset. Thus FAM64A plays an important role in determining the kinetics of mitotic progression (Zhao, et al. 2008). Although the literature indicates a putative function of FAM64A in controlling cell cycle progression, division and tumorigenesis, the impairment of cell proliferation upon FAM64A depletion was modest and not sufficient to inhibit tumor growth of xenotransplanted U937 cells in an in vivo model (Barbutti, et al. 2016). Similarly, the faster metaphase-to-anaphase transition observed in FAM64A depleted cells was not sufficient to produce any of the mitotic defects often observed in cancer cells. Rather, mitotic index, spindle assembly, chromosome congregation/segregation and cytokinesis proceeded normally in FAM64A depleted cells (Zhao, et al. 2008). FAM64A has also been described to function as a transcriptional repressor in a GAL4-based reporter gene assay (Archangelo, et al. 2006). Additionally, FAM64A was able to repress the transactivation capacity of the leukemic fusion protein PICALM/MTT10 (CALM/AF10) (Archangelo, et al. 2013). Evidence further supporting the transcriptional inhibitory properties of FAM64A were provided by Zhao and colleagues, who described the interaction between FAM64A and components of the nucleosome remodeling and deacetylase (NuRD) complex, such as MTA2, HDAC1/HDAC2 and RBBP7 / RBBP4 (RBAp46/48) (Zhao, et al. 2008). The NuRD complex is implicated in transcriptional regulation and plays a role in modifying chromatin structures to initiate and maintain gene repression. Since its discovery a number of FAM64 interacting proteins have been described (e.g. PICALM (Archangelo, et al. 2006), UHMK1 (KIS) (Archangelo, et al. 2013), PRNP (PrPC) (Satoh, et al. 2009), PTBP3 (ROD1) (Brazao, et al. 2012) and NuRD complex (Zhao, et al. 2008), shedding light on the putative function of this protein. The interaction of FAM64A with the Phosphatidylinositol Binding Clathrin Assembly Protein (PICALM) was identified in a yeast two hybrid screen (Y2H) and confirmed by GST pull-down and co-immunoprecipitation experiments for both FAM64A isoforms. The FAM64A interaction region was mapped to amino acids 221 to 294 of PICALM. Through this interaction FAM64A was able to alter the subcellular localization of PICALM. The fluorescently-tagged protein YFP-PICALM localized mainly in the cytoplasm and plasma membrane of transfected NIH3T3 cells. However, when CFP-FAM64A was co-expressed, PICALM was observed both at the cytoplasm/membrane and in the nucleus of some cells at almost equal levels (Archangelo, et al. 2006). The interaction between FAM64A and the Kinase Interacting Stathmin (KIS and UHMK1) was identified in Y2H screen and confirmed by GST pull-down, co-immunoprecipitation and co-localization experiments. KIS interacts with amino-acids 136-187 of FAM64A. Kinase assay showed that FAM64A is a substrate of KIS at serine 131 (S131). In a GAL4-based reporter gene assay KIS enhanced the transcriptional repressor activity of FAM64A, independently of phosphorylation on S131 but dependent on KIS kinase activity (Archangelo, et al. 2013). RNA immunoprecipitation sequencing (RIP-seq) described the interaction between FAM64A and ROD1 (PTBP3), an RNA-binding protein involved in nonsense-mediated decay (NMD) (Brazao, et al. 2012). Protein microarray analysis identified FAM64A as an interacting partner of the cellular prion protein (PrPC). The interaction was confirmed by co-immunoprecipitation of the overexpressed tagged-proteins (Satoh, et al. 2009).
Homology	The human FAM64A shares homology with the species described in Table 1. Table 1. Homology between the human FAM64A and other species Homo sapiens FAM64A Symbol Identity (%) Protein DNA vs. P.troglodytes FAM64A 96.6 98.6 vs. vs. M. mulatta LOC712701 91.6 95.9 vs. C. lupus FAM64A 73.3 81.2 vs. B. taurus FAM64A 83.2 85.9 vs. M. musculus Fam64a 74.8 77.5 vs. R. norvegicus Fam64a 76.7 79.1 (Source: http://www.ncbi.nlm.nih.gov/homologene/)

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