Cooperative engagement and subsequent selective displacement of SR proteins define the pre-mRNA 3D structural scaffold for early spliceosome assembly

We recently reported that serine–arginine-rich (SR) protein-mediated pre-mRNA structural remodeling generates a pre-mRNA 3D structural scaffold that is stably recognized by the early spliceosomal components. However, the intermediate steps between the free pre-mRNA and the assembled early spliceosome are not yet characterized. By probing the early spliceosomal complexes in vitro and RNA-protein interactions in vivo, we show that the SR proteins bind the pre-mRNAs cooperatively generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some of the SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65 and U2AF35. Our work thus identifies a novel function of U1 snRNP in mammalian splicing substrate definition, explains the need for excess U1 snRNP compared to other U snRNPs in vivo, demonstrates how excess SR proteins could inhibit splicing, and provides a conceptual basis to examine if this mechanism of splicing substrate definition is employed by other splicing regulatory proteins.     

Inhibition of NF-kappaB activity by IkappaBbeta in association with kappaB-Ras

IkappaBbeta, one of the major IkappaB proteins, is only partially degraded in response to most extracellular signals. However, the molecular mechanism of this event is unknown. We show here that IkappaBbeta exists in at least two different forms: one that is bound to the NF-kappaB dimer and the other bound to both NF-kappaB and kappaB-Ras, a Ras-like small G protein. Removal of cellular kappaB-Ras enhances whereas excess kappaB-Ras blocks induced IkappaBbeta degradation. Remarkably, kappaB-Ras functions in both GDP- and GTP-bound states, and mutations of the conserved guanine-binding residues of kappaB-Ras abrogate its ability to block degradation of IkappaBbeta. kappaB-Ras also directly blocks the in vitro phosphorylation of IkappaBbeta by IKKbeta. These observations suggest that IkappaBbeta in the ternary complex is resistant to degradation by most signals. We suggest that specific signals, in addition to those that activate only IKK, are essential for the complete degradation of IkappaBbeta.     

Regulation of protein kinases; controlling activity through activation segment conformation

There are currently at least forty-six unique protein kinase crystal structures, twenty-four of which are available in an active state. Here we examine these structures using a structural bioinformatics approach to understand how the conformation of the activation segment controls kinase activity.     

Mechanistic insights into Sky1p,a yeast homologue of the mammalian SR protein kinases

The SRPK family is distinguished from typical eukaryotic protein kinases by several unique structural features recently elucidated by X-ray diffraction methods [Nolen et al. (2001) Nat. Struct. Biol. 8, 176-183]. To determine whether these features impart unique catalytic function, the phosphorylation of the physiological Sky1p substrate, Npl3p, was monitored using steady-state and pre-steady-state kinetic techniques. While Sky1p has a low apparent affinity for ATP compared to other protein kinases, it binds Npl3p with very high affinity. The latter is achieved through a combination of local and distal factors in the protein substrate. The phosphoryl donor ATP has access to the nucleotide pocket in the absence or presence of Npl3p, indicating that a large protein substrate does not enforce an ordered addition of ligands. Sky1p binds two Mg(2+)-the first is essential whereas the second further enhances catalysis. While the turnover number is low (0.5 s(-1)), Npl3p is rapidly phosphorylated in the active site (40 s(-1)) based on single turnover experiments. These results indicate that Sky1p employs a catalytic pathway involving fast phosphoryl transfer followed by slow net release of products. These studies represent the first kinetic investigation of a member of the SRPK family and the first pre-steady-state kinetic study of a protein kinase using a natural protein substrate.     

The RGG domain of Npl3p recruits Sky1p through docking interactions

The SR protein kinase in yeast, Sky1p, phosphorylates yeast SR-like protein, Npl3p, at a single serine residue located at its C terminus. We report here the X-ray crystal structure of Sky1p bound to a substrate peptide and ADP. Surprisingly, an Npl3p-derived substrate peptide occupies a groove 20 A away from the kinase active site. In vitro studies support the substrate-docking role of this groove. Mutagenesis and binding studies reveal that multiple degenerate short peptide motifs located within the RGG domain of Npl3p serve as the substrate docking motifs. However, a single docking motif is sufficient for its stable interaction with the kinase. Methylation of the docking motifs abolishes kinase binding and phosphorylation of Npl3p. Remarkably, removal of the docking groove in the kinase or the docking motifs of the substrate does not reduce the overall catalytic efficiency of the phosphorylation reaction in any significant manner. We suggest that docking interaction between Sky1p and Npl3p is essential for substrate recruitment and binding specificity.     

NF-kappaB RelB forms an intertwined homodimer

The X-ray structure of the RelB dimerization domain (DD) reveals that the RelBDD assumes an unexpected intertwined fold topology atypical of other NF-kappaB dimers. All typical NF-kappaB dimers are formed by the association of two independently folded immunoglobulin (Ig) domains. In RelBDD, two polypeptides reconstruct both Ig domains in the dimer with an extra beta sheet connecting the two domains. Residues most critical to NF-kappaB dimer formation are invariant in RelB, and Y300 plays a positive role in RelBDD dimer formation. The presence of RelB-specific nonpolar residues at the surface removes several intradomain surface hydrogen bonds that may render the domain fold unstable. Intertwining may stabilize the RelBDD homodimer by forming the extra beta sheet. We show that, as in the crystal, RelB forms an intertwined homodimer in solution. We suggest that the transiently stable RelB homodimer might prevent its rapid degradation, allowing for heterodimer formation with p50 and p52.     

The 20S proteasome processes NF-kappaB1 p105 into p50 in a translation-independent manner

The NF-kappaB p50 is the N-terminal processed product of the precursor, p105. It has been suggested that p50 is generated not from full-length p105 but cotranslationally from incompletely synthesized molecules by the proteasome. We show that the 20S proteasome endoproteolytically cleaves the fully synthesized p105 and selectively degrades the C-terminus of p105, leading to p50 generation in a ubiquitin-independent manner. As small as 25 residues C-terminus to the site of processing are sufficient to promote processing in vivo. However, any p105 mutant that lacks complete ankyrin repeat domain (ARD) is processed aberrantly, suggesting that native processing must occur from a precursor, which extends beyond the ARD. Remarkably, the mutant p105 that lacks the internal region including the glycine-rich region (GRR) is completely degraded by 20S proteasome in vitro. This suggests that the GRR impedes the complete degradation of the p105 precursor, thus contributing to p50 generation.     

Identification of functionally distinct regions that mediate biological activity of the protein kinase a homolog Tpk2

Kinases regulate key signaling processes that are increasingly implicated in development and disease. Kinase modulators have become important therapeutic tools and often target catalytic domains that are among the most structurally and functionally conserved regions of these enzymes. Such therapies lose efficacy as mutations conferring resistance arise. Because interactions between distinct and often distant regions of kinases can be critical, we took an unbiased genetic approach to identify sites within the protein kinase A homolog Tpk2 that contribute to its biological activity. Because many of these map outside the conserved core, this approach should be broadly useful in identifying new, more kinase-specific therapeutic targets.