The arginine-serine (RS)-rich domain of the SR protein ASF/SF2 is phosphorylated by SR protein kinases (SRPKs) and Clk/Sty kinases. However, the mode of phosphorylation by these kinases and their coordination in the biological regulation of ASF/SF2 is unknown. Here, we report the crystal structure of an active fragment of human SRPK1 bound to a peptide derived from an SR protein. This structure led us to identify a docking motif in ASF/SF2. We find that this docking motif restricts phosphorylation of ASF/SF2 by SRPK1 to the N-terminal part of the RS domain - a property essential for its assembly into nuclear speckles. We further show that Clk/Sty causes release of ASF/SF2 from speckles by phosphorylating the C-terminal part of its RS domain. These results suggest that the docking motif of ASF/SF2 is a key regulatory element for sequential phosphorylation by SRPK1 and Clk/Sty and, thus, is essential for its subcellular localization.

SR proteins, named for their multiple arginine͞serine (RS) dipeptide repeats, are critical components of the spliceosome, influencing both constitutive and alternative splicing of pre-mRNA. SR protein function is regulated through phosphorylation of their RS domains by multiple kinases, including a family of evolutionarily conserved SR protein-specific kinases (SRPKs). The SRPK family of kinases is unique in that they are capable of phosphorylating repetitive RS domains with remarkable specificity and efficiency. Here, we carried out kinetic experiments specially developed to investigate how SRPK1 phosphorylates the model human SR protein, ASF͞SF2. By using the start-trap strategy, we monitored the progress curve for ASF͞SF2 phosphorylation in the absence and presence of an inhibitor peptide directed at the active site of SRPK1. ASF͞SF2 modification is not altered when the inhibitor peptide (trap) is added with ATP (start). However, when the trap is added first and allowed to incubate for a specific delay time, the decrease in phosphate content of the enzyme-substrate complex follows a simple exponential decline corresponding to the release rate of SRPK1. These data demonstrate that SRPK1 phosphorylates a specific region within the RS domain of ASF͞SF2 by using a fully processive catalytic mechanism, in which the splicing factor remains ''locked'' onto SRPK1 during RS domain modification.B oth the assembly of the spliceosome and the specificity of splice-site selection in mammalian cells require SR proteins, which contain one or two RNA-recognition motifs (RRMs) in the N terminus and a signature domain enriched with arginine͞ serine dipeptide repeats (RS) in the C terminus (1, 2). The SR proteins are phosphorylated and activated by the SRPK and Clk͞Sty families of protein kinases (3)(4)(5). This posttranslational modification is required for translocation of the SR protein from the cytoplasm to the nucleus (6, 7) and recruitment of the SR proteins from nuclear speckles (also known as splicing-factor compartments) to nascent transcripts for cotranscriptional splicing (8-10). Phosphorylated SR proteins are believed to facilitate 5Ј splice-site recognition through interaction with the RS domain in U1-70 K (a component of the U1 small nuclear ribonucleoprotein) and 3Ј splice-site selection by means of the RS domains present in the U2AF heterodimer (11-13). Finally, reversal of the kinase action by protein phosphatases appears to be essential for spliceosome activation and the return of splicing factors to nuclear speckles (5). ASF͞SF2 is a prototypical SR protein with two RRM domains for RNA binding and a Cterminal RS domain for mediating protein-protein interactions during spliceosome assembly (see Fig. 1C). A total of 20 serine residues lie in the RS domain and are potential sites for phosphorylation. Mutational studies have verified that phosphorylation occurs exclusively in the RS domain (3,7,14).Although the importance of phosphorylation of SR proteins is well documented for their biological functions in splici...

Highlights d High ECM stiffness activates LYN kinase to promote EMT and invasion d LYN directly phosphorylates TWIST1 to promote nuclear localization of TWIST1 d High ECM stiffness promotes ligand-independent EPHA2 signaling to activate LYN d EPHA2/LYN axis promotes breast tumor invasion and metastasis

Summary 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 serinearginine 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.

SR proteins are essential splicing factors whose biological function is regulated through phosphorylation of their C-terminal RS domains. Prior studies have shown that cytoplasmic–nuclear translocalization of the SR protein SRSF1 is regulated by multisite phosphorylation of a long Arg-Ser repeat in the N-terminus of the RS domain while subnuclear localization is controlled by phosphorylation of a shorter Arg-Ser repeat along with several Ser-Pro dipeptides in the C-terminus of the RS domain. To better understand how these two kinases partition Arg-Ser versus Ser-Pro specificities, we monitored the phosphorylation of SRSF1 by CLK1 and SRPK1. Although SRPK1 initially binds at the center of the RS domain phosphorylating in an orderly, N-terminal direction, CLK1 makes widespread contacts in the RS domain and generates multiple enzyme–substrate complexes that induce a random addition mechanism. While SRPK1 rapidly phosphorylates N-terminal serines, SRPK1 and CLK1 display similar activities toward Arg-Ser repeats in the C-terminus, suggesting that these kinases may not separate function in a strict linear manner along the RS domain. CLK1 induces a unique gel shift in SRSF1 that is not the result of enhanced Arg-Ser phosphorylation but rather is the direct result of the phosphorylation of several Ser-Pro dipeptides. These prolines are important for binding and phosphorylation of the SR protein by CLK1 but not for the SRPK1-dependent reaction. The data establish a new view of SR protein regulation in which SRPK1 and CLK1 partition activities based on Ser-Pro versus Arg-Ser placement rather than on N- and C-terminal preferences along the RS domain.

The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC 50 of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition. [Mol Cancer Ther 2009;8(12):3181-90]

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 this study, we address the effects of discrete serine-proline phosphorylation on the conformation and cellular function of the SR protein SRSF1. 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 one hundred 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 programs.

Highlights d SRPK1 phosphorylates protamine to initiate paternal genome reprogramming d Phosphorylation site mutations in protamine inhibit protamine-to-histone exchange d Phosphorylated protamine permits efficient recruitment of NPM2 and HIRA d Paternal and maternal genomes undergo synchronized reprogramming upon fertilization