Pre-mRNA splicing is regulated by developmental and environmental cues, but little is known about how specific signals are transduced in mammalian cells to regulate this critical gene expression ...step. Here, we report massive reprogramming of alternative splicing in response to EGF signaling. By blocking individual branches in EGF signaling, we found that Akt activation plays a major role, while other branches, such as the JAK/STAT and ERK pathways, make minor contributions to EGF-induced splicing. Activated Akt next branches to SR protein-specific kinases, rather than mTOR, by inducing SRPK autophosphorylation that switches the splicing kinases from Hsp70- to Hsp90-containing complexes. This leads to enhanced SRPK nuclear translocation and SR protein phosphorylation. These findings reveal a major signal transduction pathway for regulated splicing and place SRPKs in a central position in the pathway, consistent with their reputed roles in a large number of human cancers.
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► SR kinases are a major branch in the EGF pathway to regulate alternative splicing ► Activated Akt induces SRPK1autophosphorylation ► Activated SRPK1 translocates to the nucleus in an Hsp90-dependent manner ► Induced SR protein hyperphosphorylation leads to widespread changes in splicing
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The splicing of mRNA requires a group of essential factors known as SR proteins, which participate in the maturation of the spliceosome. These proteins contain one or two RNA recognition motifs and a ...C-terminal domain rich in Arg-Ser repeats (RS domain). SR proteins are phosphorylated at numerous serines in the RS domain by the SR-specific protein kinase (SRPK) family of protein kinases. RS domain phosphorylation is necessary for entry of SR proteins into the nucleus, and may also play important roles in alternative splicing, mRNA export, and other processing events. Although SR proteins are polyphosphorylated in vivo, the mechanism underlying this complex reaction has only been recently elucidated. Human alternative splicing factor serine/arginine-rich splicing factor 1 (SRSF1), a prototype for the SR protein family, is regiospecifically phosphorylated by SRPK1, a post-translational modification that controls cytoplasmic-nuclear localization. SRPK1 binds SRSF1 with unusually high affinity, and rapidly modifies about 10-12 serines in the N-terminal region of the RS domain (RS1), using a mechanism that incorporates sequential, C-terminal to N-terminal phosphorylation and several processive steps. SRPK1 employs a highly dynamic feeding mechanism for RS domain phosphorylation in which the N-terminal portion of RS1 is initially bound to a docking groove in the large lobe of the kinase domain. Upon subsequent rounds of phosphorylation, this N-terminal segment translocates into the active site, and a β-strand in RNA recognition motif 2 unfolds and occupies the docking groove. These studies indicate that efficient regiospecific phosphorylation of SRSF1 is the result of a contoured binding cavity in SRPK1, a lengthy Arg-Ser repetitive segment in the RS domain, and a highly directional processing mechanism.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Mechanical cues from the extracellular matrix (ECM) regulate various cellular processes via distinct mechanotransduction pathways. In breast cancer, increased ECM stiffness promotes ...epithelial-to-mesenchymal transition (EMT), cell invasion, and metastasis. Here, we identify a mechanosensitive EPHA2/LYN protein complex regulating EMT and metastasis in response to increasing ECM stiffness during tumor progression. High ECM stiffness leads to ligand-independent phosphorylation of ephrin receptor EPHA2, which recruits and activates the LYN kinase. LYN phosphorylates the EMT transcription factor TWIST1 to release TWIST1 from its cytoplasmic anchor G3BP2 to enter the nucleus, thus triggering EMT and invasion. Genetic and pharmacological inhibition of this pathway prevents breast tumor invasion and metastasis in vivo. In human breast cancer samples, activation of this pathway correlates with collagen fiber alignment, a marker of increasing ECM stiffness. Our findings reveal an EPHA2/LYN/TWIST1 mechanotransduction pathway that responds to mechanical signals from the tumor microenvironment to drive EMT, invasion, and metastasis.
•High ECM stiffness activates LYN kinase to promote EMT and invasion•LYN directly phosphorylates TWIST1 to promote nuclear localization of TWIST1•High ECM stiffness promotes ligand-independent EPHA2 signaling to activate LYN•EPHA2/LYN axis promotes breast tumor invasion and metastasis
Fattet et al. identified a EPHA2/LYN/TWIST1 signaling cascade activated by mechanical forces from the extracellular matrix to promote epithelial-mesenchymal transition and cell invasion. They demonstrate that activation of this mechanotransduction pathway promotes breast tumor invasion and metastasis and is involved in human breast cancer metastasis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Phosphorylation has been generally thought to activate the SR family of splicing factors for efficient splice-site recognition, but this idea is incompatible with an early observation that ...overexpression of an SR protein kinase, such as the CDC2-like kinase 1 (CLK1), weakens splice-site selection. Here, we report that CLK1 binds SR proteins but lacks the mechanism to release phosphorylated SR proteins, thus functionally inactivating the splicing factors. Interestingly, CLK1 overcomes this dilemma through a symbiotic relationship with the serine-arginine protein kinase 1 (SRPK1). We show that SRPK1 interacts with an RS-like domain in the N terminus of CLK1 to facilitate the release of phosphorylated SR proteins, which then promotes efficient splice-site recognition and subsequent spliceosome assembly. These findings reveal an unprecedented signaling mechanism by which two protein kinases fulfill separate catalytic features that are normally encoded in single kinases to institute phosphorylation control of pre-mRNA splicing in the nucleus.
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•SRPK1 and CLK1 form a stable complex in the nucleus of cells•The kinase domain of SRPK1 interacts with the disordered N terminus of CLK1•SRPK1 acts as a release factor that strips CLK1 from tightly bound SR proteins•SRPK1-induced release promotes U1 binding and mRNA splicing
Although CLK1 is required for splicing, this protein kinase does not efficiently dissociate its product, phosphorylated splicing factor SRSF1. Aubol et al. show that nuclear SRPK1 strips CLK1 from the tightly bound SRSF1, freeing the splicing factor for engagement with the U1 component of the spliceosome and splicing activation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The reversible phosphorylation of proteins ranks among the most important posttranslational modifications that occur in the cell. In the last several decades, protein phosphorylation has shifted from ...a singular curiosity to a universal mechanism for cellular control and adaptation.
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IJS, KILJ, NUK, PNG, UL, UM
Serine‐arginine protein kinase 1 (SRPK1) phosphorylates serine‐arginine (SR) proteins in the cytoplasm, directing them to the nucleus for splicing function. SRPK1 has also been detected in the ...nucleus but its function here is still not fully understood. We now demonstrate that nuclear SRPK1 can regulate U1‐70K, a protein component of the uridine‐rich 1 small nuclear ribonucleoprotein (U1 snRNP) that binds SR proteins and facilitates 5′ splice‐site selection in precursor mRNA. We found that SRPK1 uses a large, disordered domain to bind U1‐70K, regulating the interaction of an exonic splicing enhancer (ESE) to the associated SR protein. Surprisingly, the catalytic activity of SRPK1 is not required for this phenomenon. Instead, SRPK1 associates directly with the N‐terminus of U1‐70K and alters the regulatory function of the distal C‐terminus, modifying interactions between the U1‐70K:SR protein complex and the ESE. Disruption of SRPK1 binding to this complex affects the alternative splicing of genes modulated by the C‐terminus of U1‐70K. Such findings suggest that, in addition to operating as a traditional serine‐modifying catalyst, SRPK1 can also bypass this intrinsic activity to regulate RNA contacts in an early pre‐spliceosomal complex.
The 5' splice site in precursor mRNA is initially recognized by U1 snRNP (small nuclear ribonucleoprotein). This interaction is facilitated by phosphorylation‐dependent interactions between U1‐70K in U1 snRNP and members of the serine‐arginine (SR) protein family, which bind upstream RNA sequences in exons. Here, Aubol and Adams show that the serine‐arginine protein kinase SRPK1 uses a disordered region to bind U1 snRNP and modulate RNA interactions by inducing conformational changes across U1‐70K in an activity‐independent manner. This work unveils a previously unknown role of nuclear SRPK1 as a non‐catalytic, structural regulator of RNA interactions in an early pre‐spliceosomal protein complex.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
7.
Biocatalysis: A Pharma Perspective Adams, Joseph P.; Brown, Murray J. B.; Diaz‐Rodriguez, Alba ...
Advanced synthesis & catalysis,
June 6, 2019, Volume:
361, Issue:
11
Journal Article
Peer reviewed
Open access
Biocatalysis over the past few years has matured into an essential tool for modern, cost effective and sustainable pharmaceutical manufacturing. While some reaction classes are well established, and ...may even be the option of first intent, other more recently discovered enzyme classes are being rapidly developed both in academia and industry. Notwithstanding this, there are further promising enzymes that require further investment and investigation to allow their future industrial use. We here outline GSK's perspective on the current status of biocatalysis for pharmaceutical manufacturing and provide our views on areas of significant potential.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
A unique lipase (SpL) from Sphingomonas sp. HXN-200 was discovered as the first intracellular enzyme for the aminolysis of ester or acid to produce amide. Reactions of a series of esters and amines ...with SpL gave the corresponding amides 3a–g in high yield with high activity. SpL also showed high enantioselectivity and high activity for enantioselective ester aminolysis, producing amides (R)-3h–j in high ee from the corresponding racemic ester or amine. Moreover, SpL was found to be highly active for the aminolysis of carboxylic acid, which was generally considered infeasible with the known aminolysis enzymes. The aminolysis of several carboxylic acids afforded the corresponding amides 3a, 3d, 3k, 3l, and 3n in good yield. The intracellular SpL was expressed in Escherichia coli cells to give an efficient whole-cell biocatalyst for amide synthesis. Remarkably, high catalytic activity was observed in the presence of water at 2–4% (v/v) for free enzyme and 16% (v/v) for whole cells, respectively. Accordingly, E. coli (SpL) wet cells were used as easily available and practical catalysts for the aminolysis of ester or acid, producing a group of useful and valuable amides in high concentration (up to 103 mM) and high yield. The newly discovered intracellular SpL with unique properties is a promising catalyst for green and efficient synthesis of amides.
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IJS, KILJ, NUK, PNG, UL, UM
Triple mutant K66Q/S149G/N262C (TM_pheDH) of Rhodococcus phenylalanine dehydrogenase (pheDH) was engineered by directed evolution as the first enzyme for the highly enantioselective reductive ...amination of phenylacetone 1 and 4-phenyl-2-butanone 3, giving (R)-amphetamine 2 and (R)-1-methyl-3-phenylpropylamine 4 in >98% ee, respectively. The new amine dehydrogenase TM_pheDH with special substrate specificity is a valuable addition to the amine dehydrogenase family with very limited number, for asymmetric reductive amination of ketone, an important reaction in sustainable pharmaceutical manufacturing. Molecular docking provided insight into the role of key mutations of pheDH, being useful for engineering new amine dehydrogenases with higher activity and unique substrate scope.
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IJS, KILJ, NUK, PNG, UL, UM
The assembly and activation of the spliceosome rely upon the phosphorylation of an essential family of splicing factors known as the serine–arginine (SR) proteins. Although it has been demonstrated ...recently that two enzyme families, the SR protein kinases (SRPKs) and the Cdc2‐like kinases (CLKs), can function as a complex to efficiently phosphorylate these SR proteins in the nucleus, the molecular features involved in such a connection are unknown. In this study, we identified a group of conserved residues in the large lobe of SRPK1 that interact with the N terminus of CLK1 stabilizing the SRPK1‐CLK1 complex. Mutations in this motif not only disrupt formation of the kinase–kinase complex but also impair SRPK1‐dependent release of the phospho‐SR protein from CLK1. The binding motif potently up‐regulates CLK1‐specific phosphorylation sites, enhances SR protein diffusion from nuclear speckles, and impacts the alternative splicing of several target genes. These results indicate that CLK1 binds a conserved, electronegative surface on SRPK1, thereby controlling SR protein phosphorylation levels for enhanced subnuclear trafficking and alternative splicing regulation.
Splicing of mRNA is dependent on phosphorylation of an essential family of splicing factors known as SR proteins. Although protein kinases SRPK1 and CLK1 control such phosphorylation in separate cellular compartments, recent studies indicate that they can also function as a kinase–kinase complex in the nucleus. In this study, we report the discovery of a conserved docking region on SRPK1 that binds the N terminus of CLK1, supporting SR protein phosphorylation and subnuclear dynamics.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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