Identidad del Proceso Docente – Educativo de la UAM

There are three classes of splicing kinases in human which include the serine-arginine protein kinases (SRPK1/2/3), the CDC-like kinases (CLK1/2/3/4) and the pre-mRNA processing factor 4 kinases (PRP4K) with each has distinct cellular localization based on their different roles in splicing regulation (165). The PRP4K is a lesser-known splicing kinase and shown to regulate spliceosome assembly through the phosphorylation of splicing factors PRP6 and PRP31, whilst CLK1 and Serine-Arginine protein kinases (SRPKs) family are responsible for the phosphorylation of serine residues in SR protein.

SRPKs family comprises of more than 50 members that have been detected in the genomes of mammals, fungi, insects, nematode and plants (166). Most of the functional studies on mammalian SRPKs were based on SRPK1, which is one of the first protein kinases to be studied in literature, followed by SRPK2 and SRPK3 which were identified based on sequence homology with SRPK1 (167). While the functions of SRPK1 and SRPK2 are in the regulation of SR proteins distribution through phosphorylation, SRPK3 has been identified to be involved in normal muscle growth and homeostasis (167,168).

SRPK1 is a 92kDa SR protein kinase found in the cytoplasm of most cell types and tissues that act as downstream AKT target for transducing growth signal from cell surface to the nucleus (169). It is anchored in the cytoplasm by networking with chaperones and is thought to be the key splicing regulator in the alternative splicing mechanism (170). Zhong et al demonstrated that SRPK1 binds to the co-chaperones Hsp40/DNAjc8 and Aha before interacts with major molecular chaperones Hsp70 and

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Hsp90 in the cytoplasm (171). Following this, SRPK1 dissociates from these chaperone complexes which can be triggered by many factors such as stress signal, and translocates to the nucleus and initiate SR proteins phosphorylation and alter the splice site of target mRNA (171).

Cytoplasmic SRPK1 phosphorylates RS domain of serine arginine-rich (SR) proteins that induces them to relocate back to the nucleus where they can influence splice site usage for alternative splicing (172–174). Studies show that additional phosphorylation is required to recruit the already phosphorylated SR proteins in the nucleus to nascent pre-mRNA transcript and this second phosphorylation is mediated by CLK1 (175). These sequential phosphorylation events are proposed to be regulated by two factors; distinct cellular localization of the kinases (SRPK1 can be found in both cytoplasm and nucleus, CLK1 in nucleus only) and substrate specificity of the splicing kinases. For example, cytoplasmic SRPK1 phosphorylates the Arg-Ser repeats (RS1) at N- terminal of splicing protein SRSF1, whereas CLK1 phosphorylates Ser-Pro repeats (RS2) at C- terminal of SRSF1 (Figure 1.10) (173,176). Recently, studies have suggested that after binding to SR proteins to phosphorylate them, CLKs may require a release factor that can unleash them from the phosphorylated SR proteins to make it fully functional and SRPK1 is shown to function as release factor for CLK1 (Figure 1.11) (177,178). This therefore indicates that SRPK1 and CLK1 interact and work synergistically, rather that competitively, in phosphorylating RS domain of SR proteins and thus promoting spliceosome assembly in the nucleus (179).

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Figure 1.10 SRPK1 phosphorylates SR proteins in the cytoplasm hence induces SR proteins shuttling into the nucleus. CLK1 mediates additional SR proteins phosphorylation and promotes theirs release from splicing speckles to nascent pre- mRNA transcript. Once splicing completed, SR proteins bound to mRNA are dephosphorylated by nuclear phosphatases (PP1/PP2A), resulting in the recycling of the SR to the cytoplasm for the translation regulation or re-phosphorylated by SRPKs for the next round of splicing. [Adapted from Corkery et al, 2015 (165)].

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Figure 1.11 SRPK1 and CLK1 works in a cooperative manner in the nucleus of cells, where in addition to phosphorylating SR proteins, SRPK1 acts as release factor that remove CLK1 from tightly bound SR proteins and promotes U1 binding to pre-mRNA transcript thus stimulating mRNA splicing. [Adapted from Aubol et al, 2016 (179)].

Furthermore, in mammalian central nervous system, SRPK1, through the phosphorylation of SR proteins, has been shown to regulate alternative splicing events leading to the production of neuron-specific protein isoforms important in neuronal development, learning, memory and cell communication (180,181). For example, SRPK1 was demonstrated to phosphorylate SRp38 that led to the alternative splicing of glutamate receptor subunit 2 (GluR2) by promoting inclusion of flip exon in GluR2 pre- mRNA transcript (182). In addition, SRPK1 also plays role in the splicing of exon 10 in Tau protein which is implicated in the pathogenesis of Frontotemporal Dementia as well as Alzheimer disease (183). Studies also show that by decreasing the SRPK1 expression in cell will result in the decrease of phosphorylated SR proteins such as SRp20, SRp30c, 9G8, SRp40, SRp55 SRp75, Tra21 and ASF/SF2, in a dose-dependent manner (165,170,184,185). This data suggests that since phosphorylation of SR proteins by

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SRPK1 would determine the pattern of splicing, the level of SRPK1 in cells is crucial to ensure balanced level of phosphorylated and dephosphorylated SR proteins.

Although SRPK1 activity is important for regulation of various cellular functions as shown by studies in which SRPK1 knockout mice demonstrated severe aberrations of cells functioning (186), its overexpression at both protein and mRNA has been shown to promote breast and colonic tumorigenesis (172,187,188). In fact, recent data from The Cancer Genome Atlas (TCGA) reveals that the expression of SRPKs are frequently altered in cancers as shown in Table 1.1, in which high expression of SRPK1 positively correlates with tumor progression (189).

In cancer cells, SRPK1-SR proteins activity has been demonstrated to contribute to the splicing regulation of various target genes splicing such as MAP2K2 and Rac1b. Depletion of SRPK1 kinase in breast, colon and pancreatic cancer cells resulting in reduced phosphorylation of SR proteins including SRSF3, SRSF4 and SRSF6 leading to altered splicing of MAP2K2 mRNA causing it unable to phosphorylate its targets, MAPK1 and MAPK3 which eventually leading to the induction of apoptosis (170,172). It has also been shown that nuclear translocation of SRPK1 in cancer cells can be induced by upstream signals such as growth factors, that can lead to activation of signaling pathways involved in various cellular events. This is demonstrated in studies by which EGF was shown to induce cytoplasmic SRPKs translocation into the nucleus via the activation of AKT, a protein kinase that plays substantial role in cell survival, and thus triggers splicing events mediated by AKT-SRPK-SR network (190).

In addition, SRPK1 has been shown to target RBM4 in myeloid leukemia cells, thus inhibiting RBM4 binding to MCL1, a gene member of BCL2 family that regulates apoptosis in cells. This subsequently leads to exon 2 exclusion from the final mRNA transcript hence producing anti-apoptotic gene isoform in cells (165). Furthermore, increased level of MCL1 gene isoform, MCL1L has been shown to positively correlate

with SRPK1-RBM4 network, where elevated level of SRPK1 causes increased accumulation of RBM4 in the cytoplasm and thus interfere with MCL1 alternative splicing, leading to the production of anti-apoptotic protein isoform in MCF7 and MDA-

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MB-231 breast cancer cells (191,192). Consistently, SRPK1 has also been identified as one of the determinants in metastatic breast cancer as it was shown to facilitate tumor cell migration in breast cancer (193).

As studies have repeatedly identified alterations that affect splicing in diverse cancer types such as mutations in splice-site sequences and mutations in genes encoding splicing factors, manipulation of splicing might provide therapeutic benefits in cancer. For examples, splicing might be modulated by using spectrum of compounds that modulate spliceosome assembly for examples by inhibiting SF3B1 which plays essential role in initiating assembly of spliceosome components or by inhibiting the phosphorylation of SR proteins through the inhibition of CLKs and SRPKs (194).

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