Protein-Protein Interaction Inhibitor of SRPKs Alters the Splicing Isoforms of VEGF and Inhibits Angiogenesis.

Serine-arginine (SR) protein kinases (SRPKs) regulate the functions of the SR-rich splicing factors by phosphorylating multiple serines within their C-terminal arginine-serine-rich domains. Dysregulation of these phosphorylation events has been implicated in many diseases, suggesting SRPKs are potential therapeutic targets. In particular, aberrant SRPK1 expression alters the balances of proangiogenic (VEGF165) and antiangiogenic (VEGF165b) splicing isoforms of the key angiogenesis factor, vascular endothelial growth factor (VEGF), through the phosphorylation of prototypic SR protein SRSF1. Here, we report a protein-protein interaction (PPI) inhibitor of SRPKs, docking blocker of SRPK1 (DBS1), that specifically blocks a conserved substrate docking groove unique to SRPKs. DBS1 is a cell-permeable inhibitor that effectively inhibits the binding and phosphorylation of SRSF1 and subsequently switches VEGF splicing from the proangiogenic to the antiangiogenic isoform. Our findings thus provide a new direction for the development of SRPK inhibitors through targeting a unique PPI site to combat angiogenic diseases.

Attenuation of amyloid-ß generation by atypical protein kinase C-mediated phosphorylation of engulfment adaptor PTB domain containing 1 threonine 35.

Amyloid-ß (Aß) is derived from the proteolytic processing of amyloid precursor protein (APP), and the deposition of extracellular Aß to form amyloid plaques is a pathologic hallmark of Alzheimer's disease (AD). Although reducing Aß generation and accumulation has been proposed as a means of treating the disease, adverse side effects and unsatisfactory efficacy have been reported in several clinical trials that sought to lower Aß levels. Engulfment adaptor phosphotyrosine-binding (PTB) domain containing 1 (GULP1) is a molecular adaptor that has been shown to interact with APP to alter Aß production. Therefore, the modulation of the GULP1-APP interaction may be an alternative approach to reducing Aß. However, the mechanisms that regulate GULP1-APP binding remain elusive. As GULP1 is a phosphoprotein, and because phosphorylation is a common mechanism that regulates protein interaction, we anticipated that GULP1 phosphorylation would influence GULP1-APP interaction and thereby Aß production. We show here that the phosphorylation of GULP1 threonine 35 (T35) reduces GULP1-APP interaction and suppresses the stimulatory effect of GULP1 on APP processing. The residue is phosphorylated by an isoform of atypical PKC (PKCζ). Overexpression of PKCζ reduces both GULP1-APP interaction and GULP1-mediated Aß generation. Moreover, the activation of PKCζ via insulin suppresses APP processing. In contrast, GULP1-mediated APP processing is enhanced in PKCζ knockout cells. Similarly, PKC ι, another member of atypical PKC, also decreases GULP1-mediated APP processing. Intriguingly, our X-ray crystal structure of GULP1 PTB-APP intracellular domain (AICD) peptide reveals that GULP1 T35 is not located at the GULP1-AICD binding interface; rather, it immediately precedes the ß1-α2 loop that forms a portion of the binding groove for the APP helix αC. Phosphorylating the residue may induce an allosteric effect on the conformation of the binding groove. Our results indicate that GULP1 T35 phosphorylation is a mechanism for the regulation of GULP1-APP interaction and thereby APP processing. Moreover, the activation of atypical PKC, such as by insulin, may confer a beneficial effect on AD by lowering GULP1-mediated Aß production.-Chau, D. D.-L., Yung, K. W.-Y., Chan, W. W.-L., An, Y., Hao, Y., Chan, H.-Y. E., Ngo, J. C.-K., Lau, K.-F. Attenuation of amyloid-ß generation by atypical protein kinase C-mediated phosphorylation of engulfment adaptor PTB domain containing 1 threonine 35.

Distinct mechanisms govern the phosphorylation of different SR protein splicing factors.

Serine-arginine (SR) proteins are essential splicing factors containing a canonical RNA recognition motif (RRM), sometimes followed by a pseudo-RRM, and a C-terminal arginine/serine-rich (RS) domain that undergoes multisite phosphorylation. Phosphorylation regulates the localization and activity of SR proteins, and thus may provide insight into their differential biological roles. The phosphorylation mechanism of the prototypic SRSF1 by serine-arginine protein kinase 1 (SRPK1) has been well-studied, but little is known about the phosphorylation of other SR protein members. In the present study, interaction and kinetic assays unveiled how SRSF1 and the single RRM-containing SRSF3 are phosphorylated by SRPK2, another member of the SRPK family. We showed that a conserved SRPK-specific substrate-docking groove in SRPK2 impacts the binding and phosphorylation of both SR proteins, and the localization of SRSF3. We identified a nonconserved residue within the groove that affects the kinase processivity. We demonstrated that, in contrast to SRSF1, for which SRPK-mediated phosphorylation is confined to the N-terminal region of the RS domain, SRSF3 phosphorylation sites are spread throughout its entire RS domain in vitro Despite this, SRSF3 appears to be hypophosphorylated in cells at steady state. Our results suggest that the absence of a pseudo-RRM renders the single RRM-containing SRSF3 more susceptible to dephosphorylation by phosphatase. These findings suggest that the single RRM- and two RRM-containing SR proteins represent two subclasses of phosphoproteins in which phosphorylation statuses are maintained by unique mechanisms, and pose new directions to explore the distinct roles of SR proteins in vivo.

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors.