UHMK1 (U2AF homology motif kinase 1)

	Figure 1. Genomic organization, alternative splicing and protein isoforms of UHMK1. Exons are represented by numbered blue boxes and introns by the black line. The positions of the exons within the genome (NC_000001.11) are numbered. Exon joining is represented for each transcript by light green, purple and orange continuous lines; dashed lines indicate the respective protein isoform; dotted lines indicate the alternatively first exon usage. Size of the transcript variants are shown in parentheses. The transcript variant 1 codes for the longer protein (UHMK1 isoform 1, light green). The transcript variant 2 comprises an alternative first exon (light purple box), which encodes the distinct 15 amino acids N-terminal of the protein (UHMK1 isoform 2, purple). The transcript variant 3 lacks exon 7, whose excision results in a frameshift and early stop codon in exon 8. The resulting UHMK1 isoform 3 (orange), exhibits a distinct C-terminal formed by 3 aminoacids encoded by the beginning of exon 8. Sizes were scaled up, where 0.5 cm symbolizes 150 bp of exonic region (blue boxes) and 624 bp of intronic regions (black line). Number and position of aminoacids are depicted for each isoform.
Description	The UHMK1 gene is located on the chromosome 1, band q23, orientated in the plus (+) strand. The genomic locus spans 32456 base pairs (NC_000001.11), contains 8 exons and two alternative first exons.
Transcription	Three alternatively spliced transcripts of 8535, 8194 and 8446 base pairs are formed (NM_175866, NM_001184763 and NM_144624, respectively). The transcript variant 1 (NM_175866) codes for the longest protein isoform, which has 419 amino acids in length (isoform 1; NP_787062). The transcript variant 2 (NM_001184763) differs in the 5' UTR and initiates translation at the alternative start codon. The resulting protein (isoform 2; NP_0011716921) of 345 amino acids has a distinct 15 amino acids N-terminal, encoded by the alternative exon 1, and the remaining 330 amino acids encoded by exons 2-8. The transcript variant 3 (NM_144624) lacks exon 7, which results in a frame shift and early stop codon within exon 8. The encoded protein (isoform 3; NP_653225) of 344 residues, shares the first 341 amino acids (exons 1-6) with isoform 1, differing only in the last 3 amino acids at the C-terminal (Figure 1). An additional processed transcript of 3345 bp (ENST00000282169.8) retaining intron 2 (between exons 2 and 3), which does not contain an open reading frame (ORF) has been annotated for this gene. Moreover, the first 540 nucleotides of UHMK1 locus (NC_000001.11) are shared with the LOC105371497 gene, which produces a 708 bp long non-coding RNA (XR_922225.1), transcribed in the opposite direction of UHMK1 (https://www.ncbi.nlm.nih.gov/gene/127933).

	Figure 2. Diagram representing UHMK1 protein and the posttranslational modifications. UHMK1 is characterized by an N-terminal kinase core of 282 aminoacids, represented in green and a C-terminal UHM of 100 aminoacids, represented in violet. All residues described to be phosphorylated or ubiquitinated in large scale proteomic studies are depicted. Source: Phosphoproteomic databases PhosphoSitePlus (http://www.phosphosite.org). UHM: U2AF homology motif (modified from Archangelo, et al. 2013).
Description	UHMK1 is a serine/threonine kinase with calculated molecular weight of 46.5 kDa and a theoretical pI of 5.59 (PhosphoSite Plus). The primary sequence of the protein is characterized by an N-terminal kinase core (282 aminoacids) and the C-terminal U2AF homology motif (UHM), responsible for establishing protein interactions with UHM-ligand motifs (ULM), particularly present among splicing factors (Kielkopf et al., 2004; Manceau et al., 2006). UHMK1 phosphorylates preferentially proline directed serine residues on its target proteins (Maucuer et al., 2000). The lysine 54 within the N-terminal region is essential for its kinase activity and autophosphorylation activity has been observed (Boehm et al., 2002; Maucuer et al., 1997). A variety of large scale proteomic studies identified two types of posttranslational modifications within UHMK1, namely lysine-ubiquitination (K190-ub, K282-ub, K383-ub and K387-ub) and phosphorylation (Y197-p, S283-p and S290-p) as indicated at the phosphoproteomic database PhosphoSitePlus (http://www.phosphosite.org) (Figure 2).
Expression	UHMK1 is ubiquitously expressed throughout rat and human tissues, with enriched expression in the nervous system (Bieche et al., 2003; Caldwell et al., 1999; Maucuer et al., 1997). Uhmk1 mRNA is expressed during rat embryonic development and increases after birth and during the first month of brain development (Bieche et al., 2003). In the adult brain, in situ hybridization revealed remarkable expression in the substantia nigra and some sensorial and motor nuclei in the brain stem (Bieche et al., 2003). In the human brain, UHMK1 expression was detected in all regions examined, with highest levels in the deeper cortical layers. Strong expression was observed in dentate gyrus, CA1, CA3 and CA4 regions of the hippocampus, in Purkinje cells and granule cell layer of the cerebellum. No expression was detected in the white matter (Bristow et al., 2009). In the hematopoietic compartment, high levels of UHMK1 transcripts were observed in differentiated lymphocytes (CD4+, CD8+ and CD19+) compared to the progenitor enriched subpopulation (CD34+) or leukemia cell lines. UHMK1 expression was upregulated in megakaryocytic-, monocytic- and granulocytic-induced differentiation of leukemia cell lines and in erythrocytic-induced differentiation of primary CD34+ cells (Barbutti et al., 2017). Levels of UHMK1 protein are induced by mitogens. In serum starved cells, UHMK1 expression was reduced in contrast to serum stimulated cells (Boehm et al., 2002; Crook et al., 2008; Petrovic et al., 2008). UHMK1 expression increased after quiescent peripheral blood lymphocytes (PBLs) were induced to proliferate upon mitogen activation (Barbutti et al., 2017). Moreover, the amount of UHMK1 protein varies throughout the cell cycle. In synchronized cells, UHMK1 accumulates in G1 phase and decreases during S phase of the cell cycle (Archangelo et al., 2013). Little is known about the transcriptional regulation of UHMK1, which was described as direct target of the transcription factors GABP (Crook et al., 2008) and FOXM1 (Petrovic et al., 2008). The core promoter region of UHMK1 was described within -141 to -41 base pairs upstream of the transcription start site and has no consensus sequences for TATA or CCAAT boxes. Instead, it has GC-box and 3 Ets-binding sites (EBS-1, EBS-2 and EBS-3), which are essential for the promoter activity, in vitro. The regions spanning EBS-1 and EBS-2 (-103/-73 bp), and EBS-3 (-52/-42 bp) bind GABP in response to serum, leading to UHMK1 expression, cell migration and cell cycle progression of VSCM cells (Crook et al., 2008). FoxM1 binds an internal regulatory region within UHMK1 and transactivates its expression in vitro. FoxM1 appears to be essential for serum-dependent activation of UHMK1 mRNA expression, as assessed in FoxM1-/- MEF cells. It was suggested that FoxM1-induced UHMK1 expression is required for UHMK1-mediated phosphorylation and consequently degradation of CDKN1B (p27Kip1) (Petrovic et al., 2008). Furthermore, UHMK1 was described as transcriptional target of the WD repeat domain 5 (WDR5), a core component of the KMT2A (MLL) / SETD1A complex, known for its methyltransferase activity on H3 lysine 4 (H3K4). The H3K4me3 epigenetic modification correlates with gene activation, thus it is suggested that WDR5-mediated H3K4me3 at UHMK1 locus promotes its expression (Chen et al., 2015).
	Figure 3. Subcellular localization of Uhmk1. Confocal image of HeLa cells transiently transfected with plasmid expressing ha-tagged Uhmk1 (pECE-HA-Kis; Manceau et al, 2008). Ectopic Uhmk1 localizes mainly to the nucleus and to a lesser extent to the cytoplasm. The anti-Kis 3B12 antibody (Manceau et al, 2012) and Phalloidin (Invitrogen A1238) were used to detected Uhmk1 and Actin, respectively. 63x objective, zoom 2,5 x. Personal data.
Localisation	The UHMK1 protein localizes mainly to the nucleus and to a lesser extent to the cytoplasm (Boehm et al., 2002; Maucuer et al., 1997) (Figure 3). Shuttling between nucleus and cytoplasm has been described for the GFP-fused protein by fluorescence recovery after photobleaching (FRAP) (Francone et al., 2010). The kinase domain is essential for the protein nuclear localization, since deletion mutants of this domain, particularly the residues 1-211, extinguished Uhmk1 signal in immunofluorescence analysis (Manceau et al., 2008). Overexpressed ha-tagged Uhmk1 localized to the RNA granules of axon and dendrites of cortical neurons (Cambray et al., 2009). Also, a nucleolar enriched localization was observed when ha-tagged Uhmk1 was co-expressed with its GFP-fused interacting partner PIMREG (Archangelo et al., 2013).
	Figure 4. Potential functions of UHMK1. 1- UHMK1 interacts with and phosphorylates the splicing factors SF1 and SF3B1. 2- UHMK1 counteracts the inhibitory effect of p27Kip1 on cell cycle. Upon mitogenic activation, UHMK1 is upregulated and phosphorylates p27Kip1, which is exported from the nucleus and targeted for degradation by the proteasome. 3- UHMK1 impairs cell migration through negatively regulating the microtubule destabilizing protein Stathmin (STMN). UHMK1-mediated phosphorylation of STMN on S38 targets the protein for degradation. 4- UHMK1 regulates the secretory pathway in neurons and endocrine cells through its interaction with the peptidylglycine α-amidating mono-oxigenase (PAM). 5- UHMK1 interacts with components of neuronal RNA granules, such as KIF3A, NonO and eEF1A. It also associates with RNP-transported mRNAs and stimulates translation driven by the β-actin 3' UTR. 6- UHMK1 interacts with and phosphorylates the proliferation marker PIMREG, suggesting a potential role in regulating proliferation. Black arrow: represents the mitogen-dependent activation of UHMK1. Grey arrows: indicate the UHMK1-mediated phosphorylation of target proteins. Grey dotted arrows: represent the fate of the UHMK1 phosphorylated proteins targeted for degradation. P: phosphorylation; Ub: ubiquitination. Illustration was drawn using Servier Medical Art.
Function	UHMK1 was described to interact with a range of proteins, shedding light on different functions of this protein in diverse cellular processes (Figure 4). UHMK1 is the only kinase that possesses the N-terminal kinase core juxtaposed to a C-terminal U2AF homology motif (UHM) (Maucuer et al., 1997). Through the UHM motif, UHMK1 interacts with the splicing factors SF1 and SF3B1 (Manceau et al., 2008). Upon interaction, UHMK1 phosphorylates SF1, which enhances SF1 specific binding to U2AF65 and reduces the SF1-U2AF65 binding to the 3' splice site RNA (Chatrikhi et al., 2016; Manceau et al., 2006). In addition, UHMK1 expression is necessary for normal phosphorylation of SF1 in vivo (Manceau et al., 2012). The fact that UHMK1 interacts with and regulates splicing factors suggests that UHMK1 might be involved in RNA metabolism. Since UHMK1 is highly expressed in neurons, it is expected to exerts important functions in the nervous system. It was demonstrated an abnormal phosphorylation of SF1 in brain extracts of neonate Uhmk1-/- mice. Also, Uhmk1 deletion resulted in increased ratio of pre-mRNA relative to mRNA, and consequently down-regulation of brain specific genes, like cys-loop ligand-gated ion channels and metabolic enzymes. Although adult Uhmk1-/- mice did not present an obvious phenotype, animal behavior was affected. The Uhmk1-/- mice displayed locomotor hyperactivity, reduced fear conditioning and learning capacities from aversive stimuli (Manceau et al., 2012). The murine Uhmk1 was described to interact with known components of neuronal RNA granules, such as KIF3A, NONO and EEF1A1. The protein colocalizes with KIF3A kinesin in neurites and is required for neuritic outgrowth in cortical mouse neurons. Furthermore, Uhmk1 associates with RNP-transported mRNAs and stimulate translation driven by the β-actin 3' UTR, suggesting that Uhmk1 contributes to modulate translation in RNA-transporting granules as a result of local signals (Cambray et al., 2009). Still, comparison of primary cultures derived from Uhmk1-/- mice did not reveal a significant difference in neuritic arborization of cortical neurons (Manceau et al., 2012). Furthermore, a study investigating Uhmk1 action on hippocampal synaptic plasticity in mice, showed that Uhmk1 knockdown impaired spine development, altered actin dynamics, and reduced postsynaptic responsiveness. Moreover, Uhmk1 depletion resulted in decrease of the postsynaptic scaffolding protein PSD-95 and of AMPA receptor subunits. Thus Uhmk1 enhances translation of AMPA receptors and stimulates dendritic spine remodeling (Pedraza et al., 2014). Another described function of UHMK1 involves the regulation of secretory pathway in neurons and endocrine cells through its interaction with peptidylglycine α-amidating mono-oxigenase (PAM) (Alam et al., 1996). PAM cytosolic domain (CD) phosphorylation by UHMK1 (Ser-949) is required for the correct routing of this protein and consequently for its ability to affect trafficking in the regulated secretion pathway (Alam et al., 2001; Caldwell et al., 1999). Lately, it was described an intramembrane proteolysis pathway for PAM, generating a soluble fragment of the cytosolic domain (sf-CD), which accumulates in the nucleus in a phosphorylation-dependent manner, modulating the expression of genes involved in the secretory pathway. UHMK1 phosphorylates sf-CD, diminishing its localization in the nucleus and negatively regulating the expression of a subset of genes (Francone et al., 2010; Rajagopal et al., 2010). An extensively documented function of UHMK1 is its ability to positively regulate cell cycle progression through phosphorylation and inhibition of the cyclin dependent kinase inhibitor (CDKI) p27Kip1. Upon mitogenic activation, UHMK1 expression is upregulated and phosphorylates p27Kip1 on serine 10 (Ser10). As a consequence, p27Kip1 is exported from nucleus to cytoplasm, where it is targeted to the proteasome and degraded, and has no longer inhibitory effect on cell cycle. Thus, UHMK1 promotes cell cycle re-entry by inactivating p27Kip1 following growth factor stimulation (Boehm et al., 2002). Another important target of UHMK1 is the microtubule-destabilizing protein, Stathmin (Maucuer et al., 1995). UHMK1 interacts with and phosphorylates Stathmin on serine 38 (Ser38), targeting this protein to proteasome. Through negative regulation of Stathmin, UHMK1 alter microtubule dynamics and consequently impairs cell migration (Langenickel et al., 2008). UHMK1 expression is upregulated upon hematopoietic cell differentiation, thus a possible role of UHMK1 in cell differentiation was proposed (Barbutti et al., 2017). This idea was supported by the fact that UHMK1 mRNA is highly expressed in the mature brain and in terminally differentiated neural cells (Bieche et al., 2003) as well as during osteoclasts differentiation (Choi et al., 2016). The human UHMK1 shares high homology with a number of species as depicted in Table 1. PIMREG (previously known as FAM64A; CATS) is a proliferation marker shown to interact with UHMK1. The fact that UHMK1 interacts with and phosphorylates PIMREG suggests that UHMK1 regulates PIMREG function and/or localization. Nevertheless, the functional implication of this interaction remains elusive (Archangelo et al., 2013).
Homology	The human UHMK1 shares high homology with a number of species as depicted in Table 1. The human UHMK1 shares high homology with a number of species as depicted in Table 1. Table 1. Homology between the human UHMK1 and other species Homo sapiens UHMK1 Symbol Protein (% Identity) DNA(% Identity) vs. P. troglodytes vs. M. mulatta UHMK1 UHMK1 99.8 (XP_001174268) 99.8 (NP_001253697) 99.7 (XM_001174268) 99.0 (NM_001266768) vs. C. lupus UHMK1 99.8 (XP_536143) 95.8 (XM_536143) vs. B. taurus UHMK1 99.8 (NP_001192514) 95.9 (NM_001205585) vs. M. musculus Uhmk1 99.3 (NP_034763) 93.0 (NM_010633) vs. R. norvegicus Uhmk1 99.3 (NP_058989) 92.6 (NM_017293) vs. G. gallus UHMK1 88.2 (XP_015145890) 81.6 (XM_015290404) vs. D. rerio uhmk1 73.6 (NP_001070127) 69.4 (NM_001076659) (Source: http://www.ncbi.nlm.nih.gov/homologene/)

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