Drew M. Lowery

Structure and function of Polo-like kinases

Polo-like kinases play critical roles during multiple stages of cell cycle progression. All Polo-like kinases contain an N-terminal Ser/Thr kinase catalytic domain and a C-terminal region that contains one or two Polo-boxes. For Polo-like kinase 1, 2, and 3, and their homologs, the entire C-terminal region, including both Polo-boxes, functions as a single modular phosphoserine/threonine-binding domain known as the Polo-box domain (PBD). In the absence of a bound substrate, the PBD inhibits the basal activity of the kinase domain. Phosphorylation-dependent binding of the PBD to its ligands releases the kinase domain, while simultaneously localizing Polo-like kinases to specific subcellular structures. These observations suggest two different models for how the PBD integrates signals arising from other mitotic kinases to target the activated kinase towards distinct substrates. The recent X-ray crystal structures of the PBD provide insights into the structural basis for PBD function and kinase regulation. Molecular modelling of the structure of the isolated kinase domain reveals a potential basis for motif-dependent substrate specificity.

Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate

Polo‐like kinase‐1 (Plk1) phosphorylates a number of mitotic substrates, but the diversity of Plk1‐dependent processes suggests the existence of additional targets. Plk1 contains a specialized phosphoserine–threonine binding domain, the Polo‐box domain (PBD), postulated to target the kinase to its substrates. Using the specialized PBD of Plk1 as an affinity capture agent, we performed a screen to define the mitotic Plk1‐PBD interactome by mass spectrometry. We identified 622 proteins that showed phosphorylation‐dependent mitosis‐specific interactions, including proteins involved in well‐established Plk1‐regulated processes, and in processes not previously linked to Plk1 such as translational control, RNA processing, and vesicle transport. Many proteins identified in our screen play important roles in cytokinesis, where, in mammalian cells, the detailed mechanistic role of Plk1 remains poorly defined. We go on to characterize the mitosis‐specific interaction of the Plk1‐PBD with the cytokinesis effector kinase Rho‐associated coiled–coil domain‐containing protein kinase 2 (Rock2), demonstrate that Rock2 is a Plk1 substrate, and show that Rock2 colocalizes with Plk1 during cytokinesis. Finally, we show that Plk1 and RhoA function together to maximally enhance Rock2 kinase activity in vitro and within cells, and implicate Plk1 as a central regulator of multiple pathways that synergistically converge to regulate actomyosin ring contraction during cleavage furrow ingression.

Natural-like function in artificial WW domains.

Protein sequences evolve through random mutagenesis with selection for optimal fitness. Cooperative folding into a stable tertiary structure is one aspect of fitness, but evolutionary selection ultimately operates on function, not on structure. In the accompanying paper, we proposed a model for the evolutionary constraint on a small protein interaction module (the WW domain) through application of the SCA, a statistical analysis of multiple sequence alignments. Construction of artificial protein sequences directed only by the SCA showed that the information extracted by this analysis is sufficient to engineer the WW fold at atomic resolution. Here, we demonstrate that these artificial WW sequences function like their natural counterparts, showing class-specific recognition of proline-containing target peptides. Consistent with SCA predictions, a distributed network of residues mediates functional specificity in WW domains. The ability to recapitulate natural-like function in designed sequences shows that a relatively small quantity of sequence information is sufficient to specify the global energetics of amino acid interactions.

Structure and mechanism of BRCA1 BRCT domain recognition of phosphorylated BACH1 with implications for cancer.

Germline mutations in the BRCA1 tumor suppressor gene often result in a significant increase in susceptibility to breast and ovarian cancers. Although the molecular basis of their effects remains largely obscure, many mutations are known to target the highly conserved C-terminal BRCT repeats that function as a phosphoserine/phosphothreonine-binding module. We report the X-ray crystal structure at a resolution of 1.85 Å of the BRCA1 tandem BRCT domains in complex with a phosphorylated peptide representing the minimal interacting region of the DEAH-box helicase BACH1. The structure reveals the determinants of this novel class of BRCA1 binding events. We show that a subset of disease-linked mutations act through specific disruption of phospho-dependent BRCA1 interactions rather than through gross structural perturbation of the tandem BRCT domains.

Polo-Like Kinase Cdc5 Controls the Local Activation of Rho1 to Promote Cytokinesis

The links between the cell cycle machinery and the cytoskeletal proteins controlling cytokinesis are poorly understood. The small guanine nucleotide triphosphate (GTP)–binding protein RhoA stimulates type II myosin contractility and formin-dependent assembly of the cytokinetic actin contractile ring. We found that budding yeast Polo-like kinase Cdc5 controls the targeting and activation of Rho1 (RhoA) at the division site via Rho1 guanine nucleotide exchange factors. This role of Cdc5 (Polo-like kinase) in regulating Rho1 is likely to be relevant to cytokinesis and asymmetric cell division in other organisms.

Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells

Self-regulated movement of Polo-like kinase 1 to the midzone of the mitotic spindle initiates a local signaling cascade that activates the cell division machinery at the cells equator.

The NDR/LATS family kinase Cbk1 directly controls transcriptional asymmetry.

Cell fate can be determined by asymmetric segregation of gene expression regulators. In the budding yeast Saccharomyces cerevisiae, the transcription factor Ace2 accumulates specifically in the daughter cell nucleus, where it drives transcription of genes that are not expressed in the mother cell. The NDR/LATS family protein kinase Cbk1 is required for Ace2 segregation and function. Using peptide scanning arrays, we determined Cbk1′s phosphorylation consensus motif, the first such unbiased approach for an enzyme of this family, showing that it is a basophilic kinase with an unusual preference for histidine −5 to the phosphorylation site. We found that Cbk1 phosphorylates such sites in Ace2, and that these modifications are critical for Ace2′s partitioning and function. Using proteins marked with GFP variants, we found that Ace2 moves from isotropic distribution to the daughter cell nuclear localization, well before cytokinesis, and that the nucleus must enter the daughter cell for Ace2 accumulation to occur. We found that Cbk1, unlike Ace2, is restricted to the daughter cell. Using both in vivo and in vitro assays, we found that two critical Cbk1 phosphorylations block Ace2′s interaction with nuclear export machinery, while a third distal modification most likely acts to increase the transcription factors activity. Our findings show that Cbk1 directly controls Ace2, regulating the transcription factors activity and interaction with nuclear export machinery through three phosphorylation sites. Furthermore, Cbk1 exhibits a novel specificity that is likely conserved among related kinases from yeast to metazoans. Cbk1 is functionally restricted to the daughter cell, and cannot diffuse from the daughter to the mother. In addition to providing a mechanism for Ace2 segregation, these findings show that an isotropically distributed cell fate determinant can be asymmetrically partitioned in cytoplasmically contiguous cells through spatial segregation of a regulating protein kinase.

The Polo-box domain: a molecular integrator of mitotic kinase cascades and Polo-like kinase function.

No abstract yet available.

AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis.

Following genotoxic stress, cells activate a complex signalling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumour suppressor p53 lies at the heart of this DNA damage response. However, it remains incompletely understood, which signalling molecules dictate the choice between these different cellular outcomes. Here, we identify the transcriptional regulator apoptosis‐antagonizing transcription factor (AATF)/Che‐1 as a critical regulator of the cellular outcome of the p53 response. Upon genotoxic stress, AATF is phosphorylated by the checkpoint kinase MK2. Phosphorylation results in the release of AATF from cytoplasmic MRLC3 and subsequent nuclear translocation where AATF binds to the PUMA, BAX and BAK promoter regions to repress p53‐driven expression of these pro‐apoptotic genes. In xenograft experiments, mice exhibit a dramatically enhanced response of AATF‐depleted tumours following genotoxic chemotherapy with adriamycin. The exogenous expression of a phospho‐mimicking AATF point mutant results in marked adriamycin resistance in vivo. Nuclear AATF enrichment appears to be selected for in p53‐proficient endometrial cancers. Furthermore, focal copy number gains at the AATF locus in neuroblastoma, which is known to be almost exclusively p53‐proficient, correlate with an adverse prognosis and reduced overall survival. These data identify the p38/MK2/AATF signalling module as a critical repressor of p53‐driven apoptosis and commend this pathway as a target for DNA damage‐sensitizing therapeutic regimens.