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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.

The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.

Serine-arginine (SR) proteins are essential splicing factors that promote numerous steps associated with mRNA processing and whose biological function is tightly regulated through multi-site phosphorylation. In the nucleus, the cdc2-like kinases (CLKs) phosphorylate SR proteins on their intrinsically disordered Arg-Ser (RS) domains, mobilizing them from storage speckles to the splicing machinery. The CLKs have disordered N termini that bind tightly to RS domains, enhancing SR protein phosphorylation. The N termini also promote nuclear localization of CLKs, but their transport mechanism is presently unknown. To explore cytoplasmic-nuclear transitions, several classical nuclear localization sequences in the N terminus of the CLK1 isoform were identified, but their mutation had no effect on subcellular localization. Rather, we found that CLK1 amplifies its presence in the nucleus by forming a stable complex with the SR protein substrate and appropriating its NLS for transport. These findings indicate that, along with their well-established roles in mRNA splicing, SR proteins use disordered protein-protein interactions to carry their kinase regulator from the cytoplasm to the nucleus.

Splicing generates many mRNA strands from a single precursor mRNA, expanding the proteome and enhancing intracellular diversity. Both initial assembly and activation of the spliceosome require an essential family of splicing factors called serine-arginine (SR) proteins. Protein phosphatase 1 (PP1) regulates the SR proteins by controlling phosphorylation of a C-terminal arginine-serine-rich (RS) domain. These modifications are vital for the subcellular localization and mRNA splicing function of the SR protein. Although PP1 has been shown to dephosphorylate the prototype SR protein splicing factor 1 (SRSF1), the molecular nature of this interaction is not understood. Here, using NMR spectroscopy, we identified two electrostatic residues in helix α2 and a hydrophobic residue in helix α1 in the RNA recognition motif 1 (RRM1) of SRSF1 that constitute a binding surface for PP1. Substitution of these residues dissociated SRSF1 from PP1 and enhanced phosphatase activity, reducing phosphorylation in the RS domain. These effects lead to shifts in alternative splicing patterns that parallel increases in SRSF1 diffusion from speckles to the nucleoplasm brought on by regiospecific decreases in RS domain phosphorylation. Overall, these findings establish a molecular and biological connection between PP1-targeted amino acids in an RRM with the phosphorylation state and mRNA-processing function of an SR protein.

The splicing of mRNA is dependent on serine-arginine (SR) proteins that are mobilized from membrane-free, nuclear speckles to the nucleoplasm by the Cdc2-like kinases (CLKs). This movement is critical for SR protein-dependent assembly of the macromolecular spliceosome. Although CLK1 facilitates such trafficking through the phosphorylation of serine-proline dipeptides in the prototype SR protein SRSF1, an unrelated enzyme known as SR protein kinase 1 (SRPK1) performs the same function but does not efficiently modify these dipeptides in SRSF1. We now show that the ability of SRPK1 to mobilize SRSF1 from speckles to the nucleoplasm is dependent on active CLK1. Diffusion from speckles is promoted by the formation of an SRPK1-CLK1 complex that facilitates dissociation of SRSF1 from CLK1 and enhances the phosphorylation of several serine-proline dipeptides in this SR protein. Down-regulation of either kinase blocks EGF-stimulated mobilization of nuclear SRSF1. These findings establish a signaling pathway that connects SRPKs to SR protein activation through the associated CLK family of kinases.

Although phosphorylation directs serine-arginine (SR) proteins from nuclear storage speckles to the nucleoplasm for splicing function, dephosphorylation paradoxically induces similar movement, raising the question of how such chemical modifications are balanced in these essential splicing factors. In this new study, we investigated the interaction of protein phosphatase 1 (PP1) with the SR protein splicing factor (SRSF1) to understand the foundation of these opposing effects in the nucleus. We found that RNA recognition motif 1 (RRM1) in SRSF1 binds PP1 and represses its catalytic function through an allosteric mechanism. Disruption of RRM1-PP1 interactions reduces the phosphorylation status of the RS domain in vitro and in cells, redirecting SRSF1 in the nucleus. The data imply that an allosteric SR protein-phosphatase platform balances phosphorylation levels in a "goldilocks" region for the proper subnuclear storage of an SR protein splicing factor.

Field-based tibial acceleration measurements are increasingly common but sampling frequencies vary between accelerometers. The purpose of this study was to determine the minimum sampling frequency needed for reliable and accurate measurement of peak axial and resultant tibial acceleration during running in the field. Tibial acceleration was measured at 7161 Hz in 19 healthy runners on concrete and grass. Acceleration data were down sampled to approximate previously used sampling frequencies. Peak axial and resultant tibial acceleration were calculated for each sampling frequency. The within-session reliability and accuracy of peak axial and resultant tibial accelerations were evaluated using intraclass correlation coefficients, mean differences, and 95% limits of agreements. Intraclass correlation coefficients greater than.9 indicated excellent within-session reliability for both peak axial and resultant tibial acceleration measured while running on concrete and grass. Peak axial and resultant tibial accelerations were 0.5 to 1.4 g lower and minimal detectable differences were up to 0.6 g higher at 102 Hz compared with higher sampling frequencies. We recommend a minimum sampling frequency of 199 Hz for accurate and reliable measurements of peak axial and resultant tibial acceleration in the field. [ABSTRACT FROM AUTHOR]

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.

Multisite phosphorylation is required for the biological function of serine-arginine (SR) proteins, a family of essential regulators of mRNA splicing. These modifications are catalyzed by serine-arginine protein kinases (SRPKs) that phosphorylate numerous serines in arginine-serine-rich (RS) domains of SR proteins using a directional, C-to-N-terminal mechanism. The present studies explore how SRPKs govern this highly biased phosphorylation reaction and investigate biological roles of the observed directional phosphorylation mechanism. Using NMR spectroscopy with two separately expressed domains of SRSF1, we showed that several residues in the RNA-binding motif 2 interact with the N-terminal region of the RS domain (RS1). These contacts provide a structural framework that balances the activities of SRPK1 and the protein phosphatase PP1, thereby regulating the phosphoryl content of the RS domain. Disruption of the implicated intramolecular RNA-binding motif 2-RS domain interaction impairs both the directional phosphorylation mechanism and the nuclear translocation of SRSF1 demonstrating that the intrinsic phosphorylation bias is obligatory for SR protein biological function.

Phosphorylation-dependent cell communication requires enzymes that specifically recognize key proteins in a sea of similar, competing substrates. The protein kinases achieve this goal by utilizing docking grooves in the kinase domain or heterologous protein adaptors to reduce 'off pathway' targeting. We now provide evidence that the nuclear protein kinase CLK1 (cell division cycle2-like kinase 1) important for splicing regulation departs from these classic paradigms by using a novel self-association mechanism. The disordered N-terminus of CLK1 induces oligomerization, a necessary event for targeting its physiological substrates the SR protein (splicing factor containing a C-terminal RS domain) family of splicing factors. Increasing the CLK1 concentration enhances phosphorylation of the splicing regulator SRSF1 (SR protein splicing factor 1) compared with the general substrate myelin basic protein (MBP). In contrast, removal of the N-terminus or dilution of CLK1 induces monomer formation and reverses this specificity. CLK1 self-association also occurs in the nucleus, is induced by the N-terminus and is important for localization of the kinase in sub-nuclear compartments known as speckles. These findings present a new picture of substrate recognition for a protein kinase in which an intrinsically disordered domain is used to capture physiological targets with similar disordered domains in a large oligomeric complex while discriminating against non-physiological targets.

Transformer 2β1 (Tra2β1) is a splicing effector protein composed of a core RNA recognition motif flanked by two arginine-serine-rich (RS) domains, RS1 and RS2. Although Tra2β1-dependent splicing is regulated by phosphorylation, very little is known about how protein kinases phosphorylate these two RS domains. We now show that the serine-arginine protein kinase-1 (SRPK1) is a regulator of Tra2β1 and promotes exon inclusion in the survival motor neuron gene 2 (SMN2). To understand how SRPK1 phosphorylates this splicing factor, we performed mass spectrometric and kinetic experiments. We found that SRPK1 specifically phosphorylates 21 serines in RS1, a process facilitated by a docking groove in the kinase domain. Although SRPK1 readily phosphorylates RS2 in a splice variant lacking the N-terminal RS domain (Tra2β3), RS1 blocks phosphorylation of these serines in the full-length Tra2β1. Thus, RS2 serves two new functions. First, RS2 positively regulates binding of the central RNA recognition motif to an exonic splicing enhancer sequence, a phenomenon reversed by SRPK1 phosphorylation on RS1. Second, RS2 enhances ligand exchange in the SRPK1 active site allowing highly efficient Tra2β1 phosphorylation. These studies demonstrate that SRPK1 is a regulator of Tra2β1 splicing function and that the individual RS domains engage in considerable cross-talk, assuming novel functions with regard to RNA binding, splicing, and SRPK1 catalysis.

SR proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine/serine-rich) domains by two major families of protein kinases. The SRPKs (SR-specific protein kinases) efficiently phosphorylate the arginine/serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs (Cdc2-like kinases) lack a docking groove and phosphorylate both arginine/serine and serine-proline dipeptides, modifications that generate a hyperphosphorylated state important for unique SR protein-dependent splicing activities. All CLKs contain long flexible N-terminal extensions (140-300 residues) that resemble the RS domains present in their substrate SR proteins. We showed that the N-terminus in CLK1 contacts both the kinase domain and the RS domain of the SR protein SRSF1 (SR protein splicing factor 1). This interaction not only is essential for facilitating hyperphosphorylation, but also induces co-operative binding of SRSF1 to RNA. The N-terminus of CLK1 enhances the total phosphoryl contents of a panel of physiological substrates including SRSF1, SRSF2, SRSF5 and Tra2β1 (transformer 2β1) by 2-3-fold. These findings suggest that CLK1-dependent hyperphosphorylation is the result of a general mechanism in which the N-terminus acts as a bridge connecting the kinase domain and the RS domain of the SR protein.

Measurements of tibial acceleration during running must be reliable to ensure valid results and reduce errors. The purpose of this study was to determine the reliability and minimal detectable difference (MDD) of peak axial and peak resultant tibial acceleration during overground and treadmill running. The authors also compared reliability and MDDs when peak tibial accelerations were determined by averaging 5 or 10 trials. Tibial acceleration was measured during overground and treadmill running of 19 participants using a lightweight accelerometer mounted to the tibia. Peak axial and peak resultant tibial accelerations were determined for each trial. Intraclass correlation coefficients determined within-session reliability, and MDDs were also calculated. Within-session reliability was excellent for all conditions (intraclass correlation coefficients =.95–.99). The MDDs ranged from 0.6 to 1.4 g for peak axial acceleration and from 1.6 to 2.0 g for peak resultant acceleration and were lowest for peak axial tibial acceleration during overground running. Averaging 10 trials did not improve reliability compared to averaging 5 trials but did result in small reductions in MDDs. For peak axial tibial acceleration only, lower MDDs indicate that overground running may be the better option for detecting small differences. [ABSTRACT FROM AUTHOR]

The first pre-steady-state kinetics of the phosphorylation of protein kinase natural substrate is presented. Results show that the yeast homologue of the mammalian SR protein kinase, Sky1p, utilizes a catalytic pathway that involves fast phosphoryl transfer.