Regulation of SR protein kinase 1 (SRPK1) function

SRPKs were classified as “stress kinases” because they mediate the cellular stress response through their translocation to the nucleus. Nuclear SRPK1 mediates the cytotoxic effects of the drug. The redistribution of SRPK1 from the cytoplasm to the nucleus was closely related to 5-FU sensitivity. Concurrent treatment of cells with 5-FU and a selective SRPK inhibitor, diminished the cytotoxic effects of the drug. Phosphorylation of threonine 326 and serine 408 of SRPK1 was essential for nuclear translocation of the kinase. The lack of complete nuclear localization of the double-phosphomimetic mutant, may also imply that the phosphorylation of Thr326 and Ser408 was necessary but not sufficient for the nuclear translocation of SRPK1 and an additional modification(s) is (are) required. The C-terminal RGG domain of TAF15 was able to associate with SRPK1 and downregulate its activity. Furthermore, overexpression of this domain partially relocalized SRPK1 to the nucleus and resulted in hypophosphorylation of SR proteins, inhibition of splicing and inhibition of Lamin B Receptor phosphorylation. Peptides comprising the RGG repeats were also able to inhibit SRPK1 activity. The N-terminal region of PIM1L is rich in SH/SR dipeptides. The role of the SR/SH sequence is crucial for the interaction of PIM1L with SRPK1 and its absence abolishes the interaction between PIM1L and SRPK1. Conjugates with c(RGDyK) peptide had a significant effect on SRPK1 activity and in cell viability of cancer cell lines. The cellular uptake of the conjugates was achieved through endocytosis.Οι SRPKs ονομάσθηκαν ως «κινάσες του στρες» επειδή μεσολαβούν στην απόκριση του κυτταρικού στρες μέσω της μετατόπισής τους στον πυρήνα. Η πυρηνική SRPK1 σχετίστηκε στενά με την ευαισθησία στο 5-FU και μεσολαβεί στις κυτταροτοξικές δράσεις του φαρμάκου. Η ταυτόχρονη κατεργασία των κυττάρων με 5-FU και έναν εκλεκτικό αναστολέα της SRPK1, μείωσε τις κυτταροτοξικές δράσεις του φαρμάκου. Η φωσφορυλίωση της θρεονίνης 326 και της σερίνης 408 της SRPK1 ήταν απαραίτητη για την πυρηνική μετατόπιση της κινάσης. Η έλλειψη πλήρους πυρηνικής μετατόπισης του διπλού φωσφομιμητικού μεταλλάγματος, μπορεί να σημαίνει ότι η φωσφορυλίωση των Thr326 και Ser408 είναι απαραίτητη αλλά όχι επαρκής για την πυρηνική μετατόπιση της SRPK1 και μια πρόσθετη τροποποίηση ίσως, απαιτείται. Η καρβόξυ-τελική περιοχή της TAF15, που είναι πλούσια σε RGG είχε ως αποτέλεσμα τη μείωση της δραστικότητας της SRPK1. Επιπλέον, η υπερέκφραση της περιοχής αυτής οδήγησε στη μερική μετατόπιση της SRPK1 στον πυρήνα, στην υποφωσφορυλίωση των πρωτεϊνών SR, στην αναστολή του ματίσματος και στην αναστολή της φωσφορυλίωσης του υποδοχέα της Λαμίνης Β. Πεπτίδια που περιλαμβάνουν τις επαναλήψεις RGG ήταν επίσης σε θέση να αναστείλουν τη δραστικότητα της SRPK1. Η Ν-τελική περιοχή της PIM1L είναι πλούσια σε διπεπτίδια SH/SR. Ο ρόλος της αλληλουχίας SR/SH είναι κρίσιμος για την αλληλεπίδραση της PIM1L με την SRPK1 και η απουσία της καθιστά αδύνατη την αλληλεπίδραση. Οι συζευγμένοι με c(RGDyK) αναστολείς είχαν σημαντική επίδραση στη δραστικότητα της SRPK1 και στην κυτταρική βιωσιμότητα καρκινικών κυτταρικών σειρών. Η κυτταρική πρόσληψη του συζεύγματος με c(RGDyK) επιτεύχθηκε μέσω της ενδοκυττάρωσης

SR Protein Kinase 1 Inhibition by TAF15

Although SRPKs were discovered nearly 30 years ago, our understanding of their mode of regulation is still limited. Regarded as constitutively active enzymes known to participate in diverse biological processes, their prominent mode of regulation mainly depends on their intracellular localization. Molecular chaperones associate with a large internal spacer sequence that separates the bipartite kinase catalytic core and modulates the kinases’ partitioning between the cytoplasm and nucleus. Besides molecular chaperones that function as anchoring proteins, a few other proteins were shown to interact directly with SRPK1, the most-studied member of SRPKs, and alter its activity. In this study, we identified TAF15, which has been involved in transcription initiation, splicing, DNA repair, and RNA maturation, as a novel SRPK1-interacting protein. The C-terminal RGG domain of TAF15 was able to associate with SRPK1 and downregulate its activity. Furthermore, overexpression of this domain partially relocalized SRPK1 to the nucleus and resulted in hypophosphorylation of SR proteins, inhibition of splicing of a reporter minigene, and inhibition of Lamin B receptor phosphorylation. We further demonstrated that peptides comprising the RGG repeats of nucleolin, HNRPU, and HNRNPA2B1, were also able to inhibit SRPK1 activity, suggesting that negative regulation of SRPK1 activity might be a key biochemical property of RGG motif-containing proteins

SR Protein Kinase 1 Inhibition by TAF15

Although SRPKs were discovered nearly 30 years ago, our understanding of their mode of regulation is still limited. Regarded as constitutively active enzymes known to participate in diverse biological processes, their prominent mode of regulation mainly depends on their intracellular localization. Molecular chaperones associate with a large internal spacer sequence that separates the bipartite kinase catalytic core and modulates the kinases’ partitioning between the cytoplasm and nucleus. Besides molecular chaperones that function as anchoring proteins, a few other proteins were shown to interact directly with SRPK1, the most-studied member of SRPKs, and alter its activity. In this study, we identified TAF15, which has been involved in transcription initiation, splicing, DNA repair, and RNA maturation, as a novel SRPK1-interacting protein. The C-terminal RGG domain of TAF15 was able to associate with SRPK1 and downregulate its activity. Furthermore, overexpression of this domain partially relocalized SRPK1 to the nucleus and resulted in hypophosphorylation of SR proteins, inhibition of splicing of a reporter minigene, and inhibition of Lamin B receptor phosphorylation. We further demonstrated that peptides comprising the RGG repeats of nucleolin, HNRPU, and HNRNPA2B1, were also able to inhibit SRPK1 activity, suggesting that negative regulation of SRPK1 activity might be a key biochemical property of RGG motif-containing proteins

Synthesis and Anti-Angiogenic Activity of Novel c(RGDyK) Peptide-Based JH-VII-139-1 Conjugates

Peptide–drug conjugates are delivery systems for selective delivery of cytotoxic agents to target cancer cells. In this work, the optimized synthesis of JH-VII-139-1 and its c(RGDyK) peptide conjugates is presented. The low nanomolar SRPK1 inhibitor, JH-VII-139-1, which is an analogue of Alectinib, was linked to the ανβ3 targeting oligopeptide c(RGDyK) through amide, carbamate and urea linkers. The chemostability, cytotoxic and antiangiogenic properties of the synthesized hybrids were thoroughly studied. All conjugates retained mid nanomolar-level inhibitory activity against SRPK1 kinase and two out of four conjugates, geo75 and geo77 exhibited antiproliferative effects with low micromolar IC50 values against HeLa, K562, MDA-MB231 and MCF7 cancer cells. The activities were strongly related to the stability of the linkers and the release of JH-VII-139-1. In vivo zebrafish screening assays demonstrated the ability of the synthesized conjugates to inhibit the length or width of intersegmental vessels (ISVs). Flow cytometry experiments were used to test the cellular uptake of a fluorescein tagged hybrid in MCF7 and MDA-MB231 cells that revealed a receptor-mediated endocytosis process. In conclusion, most conjugates retained the inhibitory potency against SRPK1 as JH-VII-139-1 and demonstrated antiproliferative and antiangiogenic activities. Further animal model experiments are needed to uncover the full potential of such peptide conjugates in cancer therapy and angiogenesis-related diseases