P21-activated Kinase Cla4 Research Articles

Phosphorylation of Histone H4T80 Triggers DNA Damage Checkpoint Recovery

SummaryIn response to genotoxic stress, cells activate a signaling cascade known as the DNA damage checkpoint (DDC) that leads to a temporary cell cycle arrest and activation of DNA repair mechanisms. Because persistent DDC activation compromises cell viability, this process must be tightly regulated. However, despite its importance, the mechanisms regulating DDC recovery are not completely understood. Here, we identify a DNA-damage-regulated histone modification in Saccharomyces cerevisiae, phosphorylation of H4 threonine 80 (H4T80ph), and show that it triggers checkpoint inactivation. H4T80ph is critical for cell survival to DNA damage, and its absence causes impaired DDC recovery and persistent cell cycle arrest. We show that, in response to genotoxic stress, p21-activated kinase Cla4 phosphorylates H4T80 to recruit Rtt107 to sites of DNA damage. Rtt107 displaces the checkpoint adaptor Rad9, thereby interrupting the checkpoint-signaling cascade. Collectively, our results indicate that H4T80ph regulates DDC recovery.

The Botrytis cinerea PAK kinase BcCla4 mediates morphogenesis, growth and cell cycle regulating processes downstream of BcRac.

Rac proteins are involved in a variety of cellular processes. Effector proteins that interact with active Rac convey the GTPase-generated signal to downstream developmental cascades and processes. Here we report on the analysis of the main effector and signal cascade downstream of BcRac, the Rac homolog of the grey mold fungus Botrytis cinerea. Several lines of evidence highlighted the p21-activated kinase Cla4 as an important effector of Rac in fungi. Analysis of Δbccla4 strains revealed that the BcCla4 protein was sufficient to mediate all of the examined BcRac-driven processes, including hyphal growth and morphogenesis, conidia production and pathogenicity. In addition, the Δbccla4 strains had altered nuclei content, a phenomenon that was previously observed in Δbcrac isolates, thus connecting the BcRac/BcCla4 module with cell cycle control. Further analyses revealed that BcRac/BcCla4 control mitotic entry through changes in phosphorylation status of the cyclin dependent kinase BcCdk1. The complete cascade includes the kinase BcWee1, which is downstream of BcCla4 and upstream of BcCdk1. These results provide a mechanistic insight on the connection of cell cycle, morphogenesis and pathogenicity in fungi, and position BcCla4 as the most essential effector and central regulator of all of these processes downstream of BcRac.

Small GTPase Rac1 and its interaction partner Cla4 regulate polarized growth and pathogenicity in Verticillium dahliae

Rac1 is a small GTPase coordinating diverse cellular functions such as cell polarity, vesicular trafficking, the cell cycle and transcriptional dynamics in many organisms. In this study, we investigate the biological functions of VdRac1, a Rac1 homolog in the soil-borne, wilt-causing fungus Verticillium dahliae. The VdRac1 gene was deleted in a V. dahliae virulence strain Vd8 isolated from a local cotton cultivar. ΔVdrac1 mutants display drastic reduction in colony expansion and form compact, convoluted colonies, show hyper-branching, loss of polarity and ability to penetrate, leading to severely reduced virulence. The p21-activated kinase Cla4 (named as VdCla4 in V. dahliae) null mutants ΔVdcla4 share identical phenotypes with ΔVdrac1. Yeast two-hybrid studies prove that VdCla4 is an effector of VdRac1. Localizations of actin and reactive oxygen species (ROS) in ΔVdrac1 and ΔVdcla4 compared with the corresponding wild-type strain reveal that VdRac1 and VdCla4 play a primary role in polarized hyphal growth via organization of ROS and play only a minor role in the organization of actin. The Vdrac1 and Vdcla4 null mutants are defective in conidiation and trace elements can partially compensate for the defect. Our data demonstrate that VdRac1 regulates polarized growth and pathogenicity by interacting with its effector VdCla4 in V. dahliae.

Cdk and the anillin homolog Bud4 define a new pathway regulating septin organization in yeast

In budding yeast, the cortical structure formed by the septins is remodeled at the onset of mitotic exit and delineates a specialized compartment dedicated to cytokinesis. How this septin function is spatially and timely regulated remains poorly understood. In this study, we report a role of the anillin-like protein Bud4 in the formation and the disassembly of the double ring structure formed by the septins at the time of cytokinesis. Bud4 acts with Bud3 in this pathway and in parallel with septin phosphorylation by the p21-activated kinase Cla4 and the septin-dependent kinase Gin4. In addition, we show that the function of Bud4 is regulated by the cyclin-dependent protein kinase Cdk1, the master regulator of cell cycle progression. This result suggests that the Cdks, or a locally specific pool of the kinase, may have a role past mitotic exit.

Solution Structure of a Novel Cdc42 Binding Module of Bem1 and Its Interaction with Ste20 and Cdc42

Bem1 is a scaffold protein essential for the establishment of cell polarity in Saccharomyces cerevisiae. This work reports the solution structure of a Cdc42 binding module of Bem1 comprising the second SH3 domain (SH3b) and its C-terminal flanking region termed Cdc42 interacting (CI). First, the structure of Bem1 SH3b-CI was determined by NMR spectroscopy, which shows that Bem1 SH3b-CI is a structurally and functionally related domain that binds Cdc42. Next, the solution structure of Bem1 SH3b-CI in complex with the proline-rich region of p21-activated kinase Ste20 (Ste20 PRR) was determined. Finally, the interaction surface of Bem1 SH3b-CI with Cdc42 was identified based on chemical shift perturbation studies which reveals that Bem1 SH3b-CI interacts simultaneously with both Ste20 PRR and Cdc42 using the opposite surfaces. Thus, Bem1 can tether Cdc42 and Ste20 in close proximity so that Cdc42 can efficiently interact with Ste20 Cdc42 and Rac interactive binding (CRIB). Based on the present results together with the previous biochemical studies (Lamson, R. E., Winters, M. J., and Pryciak, P. M. (2002) Mol. Cell. Biol. 22, 2939-2951 and Winters, M. J., and Pryciak, P. M. (2005) Mol. Cell. Biol. 25, 2177-2190), a model was suggested that the autoinhibition of Ste20 kinase activity by CRIB is released through the Cdc42-CRIB interaction, which is mediated by Bem1, and Ste20 is subsequently activated, an initial step for the establishment of the cell polarity.

The Rho GDI Rdi1 Regulates Rho GTPases by Distinct Mechanisms

The small guanosine triphosphate (GTP)-binding proteins of the Rho family are implicated in various cell functions, including establishment and maintenance of cell polarity. Activity of Rho guanosine triphosphatases (GTPases) is not only regulated by guanine nucleotide exchange factors and GTPase-activating proteins but also by guanine nucleotide dissociation inhibitors (GDIs). These proteins have the ability to extract Rho proteins from membranes and keep them in an inactive cytosolic complex. Here, we show that Rdi1, the sole Rho GDI of the yeast Saccharomyces cerevisiae, contributes to pseudohyphal growth and mitotic exit. Rdi1 interacts only with Cdc42, Rho1, and Rho4, and it regulates these Rho GTPases by distinct mechanisms. Binding between Rdi1 and Cdc42 as well as Rho1 is modulated by the Cdc42 effector and p21-activated kinase Cla4. After membrane extraction mediated by Rdi1, Rho4 is degraded by a novel mechanism, which includes the glycogen synthase kinase 3beta homologue Ygk3, vacuolar proteases, and the proteasome. Together, these results indicate that Rdi1 uses distinct modes of regulation for different Rho GTPases.

The small GTPase Rac and the p21‐activated kinase Cla4 in Claviceps purpurea: interaction and impact on polarity, development and pathogenicity

Claviceps purpurea, the ergot fungus, is a highly specialized pathogen of grasses; its colonization of host ovarian tissue requires an extended period of strictly polarized, oriented growth towards the vascular tissue. To understand this process, we study the role of signalling factors affecting polarity and differentiation. We showed that the small GTPase Cdc42 is involved in polarity, sporulation and in planta growth in C. purpurea. Here we present evidence that the GTPase Rac has an even stronger and, in some aspects, inverse impact on growth and development: Deltarac mutants form coralline-like colonies, show hyper-branching, loss of polarity, sporulation and ability to penetrate. Functional analyses and yeast two-hybrid studies prove that the p21-activated kinase Cla4 is a major downstream partner of Rac. Phosphorylation assays of MAP kinases and expression studies of genes encoding reactive oxygen species (ROS)-scavenging and -generating enzymes indicate a function of Rac and Cla4 in fungal ROS homoeostasis which could contribute to their drastic impact on differentiation.