N-terminus of the protein kinase CLK1 induces SR protein hyperphosphorylation.

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.

Partitioning RS domain phosphorylation in an SR protein through the CLK and SRPK protein kinases.

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.

Nucleotide release sequences in the protein kinase SRPK1 accelerate substrate phosphorylation.

Protein kinases are essential signaling enzymes that transfer phosphates from bound ATP to select amino acids in protein targets. For most kinases, the phosphoryl transfer step is highly efficient, while the rate-limiting step for substrate processing involves slow release of the product ADP. It is generally thought that structural factors intrinsic to the kinase domain and the nucleotide-binding pocket control this step and consequently the efficiency of protein phosphorylation for these cases.

The Akt-SRPK-SR axis constitutes a major pathway in transducing EGF signaling to regulate alternative splicing in the nucleus.

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.

Regulating SR protein phosphorylation through regions outside the kinase domain of SRPK1.

SR proteins (splicing factors containing arginine-serine repeats) are essential splicing factors whose phosphorylation by the SR-specific protein kinase (SRPK) family regulates nuclear localization and mRNA processing activity. In addition to an N-terminal extension with unknown function, SRPKs contain a large, nonhomologous spacer insert domain (SID) that bifurcates the kinase domain and anchors the kinase in the cytoplasm through interactions with chaperones. While structures for the kinase domain are now available, constructs that include regions outside this domain have been resistant to crystallographic elucidation.

Discrete retardance second harmonic generation ellipsometry.

A new instrument was constructed to perform discrete retardance nonlinear optical ellipsometry (DR-NOE). The focus of the design was to perform second harmonic generation NOE while maximizing sample and application flexibility and minimizing data acquisition time. The discrete retardance configuration results in relatively simple computational algorithms for performing nonlinear optical ellipsometric analysis.

Direct determination of effective interfacial optical constants by nonlinear optical null ellipsometry of chiral films.

Nonlinear optical null ellipsometry (NONE) measurements of chiral interfaces allowed direct experimental measurement of the linear interfacial optical constants in surface second harmonic generation (SHG) measurements. Since phase information is retained in NONE measurements, the real and imaginary components of the interfacial refractive index (n and k, respectively) were uniquely obtained from the measured chiral chi((2)) tensor elements of a fluorescein-labeled bovine serum albumin film. The sensitivity of the calculated chi((2)) tensor elements on the assumed values of the interfacial optical constants allowed measurements of n and k to four significant figures with no additional adjustable parameters and independent of molecular symmetry.

Ellipsometric approach for the real-time detection of label-free protein adsorption by second harmonic generation.

Second harmonic generation (SHG) was performed using a novel ellipsometric detection approach to selectively probe the real-time surface binding kinetics of an unlabeled protein. The coherence of nonlinear optical processes introduces new possibilities for exploiting polarization that are unavailable with incoherent methods, such as absorbance and fluorescence. Adsorption of bovine serum albumin (BSA) at silica/aqueous solution interfaces resulted in changes in the polarization state of the frequency-doubled light through weak, dynamic interactions with a coadsorbed nonlinear optical probe molecule (rhodamine 6G).