Mitogen-activated 3p kinase is active in the nucleus

The MAPK-activated kinase 3pK (chromosome 3p kinase), also known as MAPKAPK-3, is a member of a family of kinases that are activated by more than one mitogen-activated protein kinase (MAPK). 3pK is unique since it was shown to be activated by three members of the MAPK family, namely extracellular-signal-regulated kinase (ERK), p38, and Jun-N-terminal kinase (JNK). Accordingly, 3pK is highly activated both by mitogens and by stress-inducing agents or proinflammatory cytokines. Studies utilizing dominant interfering mutants and pharmacological agents revealed that upon mitogenic stimulation, 3pK is exclusively activated via the classical MAPK cascade, while stress-induced activation of 3pK is mainly mediated by p38. The mechanism defining the specificity of kinase action in response to mitogenic versus stress activation remains unknown. Here we show that 3pK is transported to the cytoplasm upon both stress and mitogenic stimulation. While kinetics of nuclear export are similar in both situations, the activation pattern differs substantially. In the mitogenic situation, active 3pK remains in the nucleus for a significant time and there may fulfill mitogen-specific functions. These data not only show that nuclear export of the kinase is mechanistically uncoupled from its activation, but also provide a novel mechanism by which cells may modulate enzyme activity toward a stimulus-specific response.     

Structural and energetic analysis of activation by a cyclic nucleotide binding domain

MlotiK1 is a prokaryotic homolog of cyclic-nucleotide-dependent ion channels that contains an intracellular C-terminal cyclic nucleotide binding (CNB) domain. X-ray structures of the CNB domain have been solved in the absence of ligand and bound to cAMP. Both the full-length channel and CNB domain fragment are easily expressed and purified, making MlotiK1 a useful model system for dissecting activation by ligand binding. We have used X-ray crystallography to determine three new MlotiK1 CNB domain structures: a second apo configuration, a cGMP-bound structure, and a second cAMP-bound structure. In combination, the five MlotiK1 CNB domain structures provide a unique opportunity for analyzing, within a single protein, the structural differences between the apo state and the bound state, and the structural variability within each state. With this analysis as a guide, we have probed the nucleotide selectivity and importance of specific residue side chains in ligand binding and channel activation. These data help to identify ligand-protein interactions that are important for ligand dependence in MlotiK1 and, more globally, in the class of nucleotide-dependent proteins.     

2′-5′-Oligoadenylate synthetase is activated by a specific RNA sequence motif

2′-5′-Oligoadenylate synthetase plays a central role in the cellular innate antiviral response. Although activation of 2′-5′-oligoadenylate synthetase by double stranded RNA was discovered more than 30 years ago it is still unclear which sequence features are required by an RNA to activate the enzyme. A pool of chemically synthesized short double stranded RNAs of specific sequence was used to probe 2′-5′-oligoadenylate synthetase activation. It was found that activating double stranded RNAs contain the following motif: NNWWNNNNNNNNNWGN. Verification of this sequence motif in a pool of 102 small double stranded RNAs demonstrated a false positive prediction rate of 8% and a false negative prediction rate of 12%. The sequence motif identified provides mechanistic insight into the mechanism of 2′-5′-oligoadenylate synthetase activation by double stranded RNA and allows theoretical predictions whether a given RNA molecule has the capability to activate 2′-5′-oligoadenylate synthetase.     

Importance of the A-helix of the catalytic subunit of cAMP-dependent protein kinase for stability and for orienting subdomains at the cleft interface.

All eukaryotic protein kinases share a conserved catalytic core. In the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) this core is preceded by a myristylation motif followed by a long helix with Trp 30 at the end of this A-helix filling a hydrophobic cavity between the two lobes of the core. To understand the importance of the A-helix, the myristylation motif (delta 1-14) as well as the entire N-terminal segment (delta 1 -39) were deleted. In addition, Trp 30 was replaced with both Tyr and Ala. All proteins were overexpressed in E. coli and purified to homogeneity. rC(delta 1-14), rC(W30Y), and rC(W30A) all had reduced thermostability, but were catalytically indistinguishable from wild-type C. Based on Surface Plasmon Resonance, all three also formed stable holoenzyme complexes with the RI-subunit, although the appKds were reduced by more than 10-fold due to decrease in the association rate. Surprisingly, however, the holoenzymes were even more thermostable than wild-type holoenzyme. To obtain active enzyme, it was necessary to purify rC(delta 1-39) as a fusion protein with glutathione-S-transferase (GST-rC(delta 1-39), although its thermostability (Tm) was decreased by 12.5 degrees C, was catalytically similar to wild-type C and was inhibited by both the type I and II R-subunits and the heat-stable protein kinase inhibitor (PKI). The Tm for holoenzyme II formed with GST-rC(delta 1-39) was 16.5 degrees C greater than the Tm for free GST-rC(delta 1-39), and the Ka(cAMP) was increased nearly 10-fold. These mutants point out striking and unanticipated differences in how the RI and RII subunits associate with the C-subunit to form a stable holoenzyme and indicate, furthermore, that this N-terminal segment, far from the active site cleft, influences those interactions. The importance of the A-helix and Trp 30 for stability correlates with its location at the cleft interface where it orients the C-helix in the small lobe and the activation loop in the large so that these subdomains are aligned in a way that allows for correct configuration of residues at the active site. This extensive network of contacts that links the A-helix directly to the active site in cAPK is compared to other kinases whose crystal structures have been solved.