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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]

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]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Results suggest that the yeast SR protein kinase, Sky1p, strongly destabilizes the gamma phosphate of ATP as determined by the affinities of several nucleotides to Sky1p, thus differeing from the cAMP-dependent protein kinase. Data indicate that both protein kinases may facilitate rapid phosphoryl transfer by a novel mechanism.

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. [ABSTRACT FROM AUTHOR]

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 selfassociation 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. [ABSTRACT FROM AUTHOR]

The alternative splicing of human genes is dependent on SR proteins, a family of essential splicing factors whose name derives from a signature C-terminal domain rich in arginine-serine dipeptide repeats (RS domains). Although the SRPKs (SR-specific protein kinases) phosphorylate these repeats, RS domains also contain prolines with flanking serines that are phosphorylated by a second family of protein kinases known as the CLKs (Cdc2-like kinases). The role of specific serine-proline phosphorylation within the RS domain has been difficult to assign since CLKs also phosphorylate arginine-serine dipeptides and, thus, display overlapping residue specificities with the SRPKs. In the present study, we address the effects of discrete serine-proline phosphorylation on the conformation and cellular function of the SR protein SRSF1 (SR protein splicing factor 1). Using chemical tagging and dephosphorylation experiments, we show that modification of serine-proline dipeptides broadly amplifies the conformational ensemble of SRSF1. The induction of these new structural forms triggers SRSF1 mobilization in the nucleus and alters its binding mechanism to an exonic splicing enhancer in precursor mRNA. These physical events correlate with changes in the alternative splicing of over 100 human genes based on a global splicing assay. Overall, these studies draw a direct causal relationship between a specific type of chemical modification in an SR protein and the regulation of alternative gene splicing programmes. [ABSTRACT FROM AUTHOR]

SR proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine/serinerich) 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 Tra2ß1 (transformer 2ß1) 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. [ABSTRACT FROM AUTHOR]

The SRPK family of protein kinases regulates mRNA splicing by phosphorylating an essential group of factors known as SR proteins, so named for a C-terminal domain enriched in arginine-serine dipeptide repeats (RS domains). SRPKs phosphorylate RS domains at numerous sites altering SR protein subcellular localization and splicing function. The RS domains in these splicing factors differ considerably in overall length and dipeptide layout. Despite their importance, little is known about how these diverse RS domains interact with SRPKs and regulate SR protein phosphorylation. We now show that sequences distal to the SRPK1 consensus region in the RS domain of the prototype SR protein SRSF1 are not passive as originally thought but rather play active roles in accelerating phosphorylation rates. Located in the C-terminal end of the RS domain, this nonconsensus region up-regulates rate-limiting ADP release through the nucleotide release factor, a structural module in SRPK1 composed of two noncontiguous sequence elements outside the kinase core domain. The data show that the RS domain in SRSF1 is multifunctional and that sequences once thought to be catalytically silent can be recruited to enhance the efficiency of SR protein phosphorylation. [ABSTRACT FROM AUTHOR]

The splicing function of SR proteins is regulated by multisite phosphorylation of their C-terminal RS (arginine-serine rich) domains. SRPK1 has been shown to phosphorylate the prototype SR protein SRSF1 using a directional mechanism in which 11 serines flanked by arginines are sequentially fed from a docking groove in the large lobe of the kinase domain to the active site Although this process is expected to operate on lengthy arginine-serine repeats (≥8), many SR proteins contain smaller repeats of only 1-4 dipeptides, raising the question of how alternate KS domain configurations are phosphorylated. To address this, we studied a splice variant of Tra2β that contains a C-terminal RS domain with short arginine-serine repeats [Tra2β)(ΔN)]. We showed that SRPK1 selectively phosphorylates several serines near the C-terminus of the KS domain. SRPK1 uses a distributive mechanism for Tra2β)(ΔN) where the rate-limiting step is the dissociation of the protein substrate rather than nucleotide exchange as in the case of SRSF1. Although a functioning docking groove is required for efficient SRSF1 phosphorylation, this conserved structural element is dispensable for Tra2β(ΔN) phosphorylation. These large shifts in mechanism are likely to account for the slower net turnover rate of Tra2β(ΔN) compared to SRSF1 and may signal fundamental differences in phosphorylation among Sit. proteins with distinctive arginine-serine profiles. Overall, these data indicate that SRPK1 conforms to changes in KS domain architecture using a flexible kinetic mechanism and selective usage of a conserved docking groove. [ABSTRACT FROM AUTHOR]