Polo-like kinases (Plks) perform crucial functions in cell-cycle progression and multiple stages of mitosis. Plks are characterized by a C-terminal noncatalytic region containing two tandem Polo boxes, termed the Polo-box domain (PBD), which has recently been implicated in phosphodependent substrate targeting. We show that the PBDs of human, Xenopus, and yeast Plks all recognize similar phosphoserine/threonine-containing motifs. The 1.9 A X-ray structure of a human Plk1 PBD-phosphopeptide complex shows that the Polo boxes each comprise beta6alpha structures that associate to form a 12-stranded beta sandwich domain. The phosphopeptide binds along a conserved, positively charged cleft located at the edge of the Polo-box interface. Mutations that specifically disrupt phosphodependent interactions abolish cell-cycle-dependent localization and provide compelling phenotypic evidence that PBD-phospholigand binding is necessary for proper mitotic progression. In addition, phosphopeptide binding to the PBD stimulates kinase activity in full-length Plk1, suggesting a conformational switching mechanism for Plk regulation and a dual functionality for the PBD.
The timing and localization of events during mitosis is controlled by the regulated phosphorylation of proteins by the mitotic kinases, which include Aurora A, Aurora B, Nek2, Plk1, and the cyclin-dependent kinase complex Cdk1/cyclin B. Although mitotic kinases can have overlapping subcellular localizations, each kinase appears to phosphorylate its substrates on distinct sites. To gain insight into the relative importance of local sequence context in kinase selectivity, identify previously unknown substrates of these five mitotic kinases, and explore potential mechanisms for substrate discrimination, we determined the optimal substrate motifs of these major mitotic kinases by Positional Scanning Oriented Peptide Library Screening (PS-OPLS). We verified individual motifs with in vitro peptide kinetic studies and used structural modeling to rationalize the kinase-specific selection of key motif-determining residues at the molecular level. Cross comparisons among the phosphorylation site selectivity motifs of these kinases revealed an evolutionarily conserved mutual exclusion mechanism in which the positively and negatively selected portions of the phosphorylation motifs of mitotic kinases, together with their subcellular localizations, result in proper substrate targeting in a coordinated manner during mitosis.
The dimeric Ser/Thr kinase Nek2 regulates centrosome cohesion and separation through phosphorylation of structural components of the centrosome, and aberrant regulation of Nek2 activity can lead to aneuploid defects characteristic of cancer cells. Mutational analysis of autophosphorylation sites within the kinase domain identified by mass spectrometry shows a complex pattern of positive and negative regulatory effects on kinase activity that are correlated with effects on centrosomal splitting efficiency in vivo. The 2.2-Å resolution x-ray structure of the Nek2 kinase domain in complex with a pyrrole-indolinone inhibitor reveals an inhibitory helical motif within the activation loop. This helix presents a steric barrier to formation of the active enzyme and generates a surface that may be exploitable in the design of specific inhibitors that selectively target the inactive state. Comparison of this "auto-inhibitory" conformation with similar arrangements in cyclin-dependent kinase 2 and epidermal growth factor receptor kinase suggests a role for dimerization-dependent allosteric regulation that combines with autophosphorylation and protein phosphatase 1c phosphatase activity to generate the precise spatial and temporal control required for Nek2 function in centrosomal maturation.Protein kinase activity is crucial for the precise regulation of the eukaryotic cell cycle. Although cyclin-dependent kinases remain the master regulators of cell cycle progression, it is clear that a variety of other proteins kinases also play important roles.Nek2 is a member of the NIMA 6 -related kinase (or Nek) family of serine/threonine protein kinases, so named because all are related to the NIMA kinase of Aspergillus nidulans (for review, see Ref. 1). In Aspergillus, NIMA is essential for mitotic entry with temperature-sensitive nimA mutations leading to cells that are "never in mitosis" (2). Like Aspergillus, other fungi also express a single NIMA-related kinase, e.g. Kin3 in Saccharomyces cerevisiae and Fin1 in Schizosaccharomyces pombe. Although the yeast Neks do not appear to be essential for cell cycle progression, elegant studies on Fin1 have revealed key functions in chromosome segregation and mitotic exit (3, 4). In contrast, higher eukaryotes express multiple Neks with 11 genes (Nek1 to Nek11) encoded within the human genome (1). With the exception of human Nek6 and Nek7 and Nek10, all Neks have an N-terminal kinase domain followed by a C-terminal non-catalytic regulatory domain. However, the length and composition of motifs present within these C-terminal extensions vary considerably, reflecting diverse roles in cell regulation.Of the human proteins, Nek2 is the most closely related to the fungal kinases, being 47% identical to NIMA within the amino acid sequence of their catalytic domains. Nek2 localizes to centrosomes. Here, it contributes to spindle pole formation during mitosis (5) through phosphorylation of proteins involved in centriolar cohesion, including C-Nap1 and rootletin, to allow spindle pole separation (6 ...
The IKK [IκB (inhibitory κB) kinase] complex is a key regulatory component of NF-κB (nuclear factor κB) activation and is responsible for mediating the degradation of IκB, thereby allowing nuclear translocation of NF-κB and transcription of target genes. NEMO (NF-κB essential modulator), the regulatory subunit of the IKK complex, plays a pivotal role in this process by integrating upstream signals, in particular the recognition of polyubiquitin chains, and relaying these to the activation of IKKα and IKKβ, the catalytic subunits of the IKK complex. The oligomeric state of NEMO is controversial and the mechanism by which it regulates activation of the IKK complex is poorly understood. Using a combination of hydrodynamic techniques we now show that apo-NEMO is a highly elongated, dimeric protein that is in weak equilibrium with a tetrameric assembly. Interaction with peptides derived from IKKβ disrupts formation of the tetrameric NEMO complex, indicating that interaction with IKKα and IKKβ and tetramerization are mutually exclusive. Furthermore, we show that NEMO binds to linear di-ubiquitin with a stoichiometry of one molecule of di-ubiquitin per NEMO dimer. This stoichiometry is preserved in a construct comprising the second coiled-coil region and the leucine zipper and in one that essentially spans the full-length protein. However, our data show that at high di-ubiquitin concentrations a second weaker binding site becomes apparent, implying that two different NEMO–di-ubiquitin complexes are formed during the IKK activation process. We propose that the role of these two complexes is to provide a threshold for activation, thereby ensuring sufficient specificity during NF-κB signalling.
Leucine zippers are oligomerization domains used in a wide range of proteins. Their structure is based on a highly conserved heptad repeat sequence in which two key positions are occupied by leucines. The leucine zipper of the cell cycle-regulated Nek2 kinase is important for its dimerization and activation. However, the sequence of this leucine zipper is most unusual in that leucines occupy only one of the two hydrophobic positions. The other position, depending on the register of the heptad repeat, is occupied by either acidic or basic residues. Using NMR spectroscopy, we show that this leucine zipper exists in two conformations of almost equal population that exchange with a rate of 17 s−1. We propose that the two conformations correspond to the two possible registers of the heptad repeat. This hypothesis is supported by a cysteine mutant that locks the protein in one of the two conformations. NMR spectra of this mutant showed the predicted 2-fold reduction of peaks in the 15N HSQC spectrum and the complete removal of cross peaks in exchange spectra. It is possible that interconversion of these two conformations may be triggered by external signals in a manner similar to that proposed recently for the microtubule binding domain of dynein and the HAMP domain. As a result, the leucine zipper of Nek2 kinase is the first example where the frameshift of coiled-coil heptad repeats has been directly observed experimentally.
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