Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing.

The effectiveness of checkpoint kinase 1 (Chk1) inhibitors at killing cancer cells is considered to be fully dependent on their effect on DNA replication initiation. Chk1 inhibition boosts origin firing, presumably limiting the availability of nucleotides and in turn provoking the slowdown and subsequent collapse of forks, thus decreasing cell viability. Here we show that slow fork progression in Chk1-inhibited cells is not an indirect effect of excess new origin firing. Instead, fork slowdown results from the accumulation of replication barriers, whose bypass is impeded by CDK-dependent phosphorylation of the specialized DNA polymerase eta (Polη). Also in contrast to the linear model, the accumulation of DNA damage in Chk1-deficient cells depends on origin density but is largely independent of fork speed. Notwithstanding this, origin dysregulation contributes only mildly to the poor proliferation rates of Chk1-depleted cells. Moreover, elimination of replication barriers by downregulation of helicase components, but not their bypass by Polη, improves cell survival. Our results thus shed light on the molecular basis of the sensitivity of tumors to Chk1 inhibition.

Cyclin Kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells.

The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21's PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis.

The fork and the kinase: a DNA replication tale from a CHK1 perspective.

Replication fork progression is being continuously hampered by exogenously introduced and naturally occurring DNA lesions and other physical obstacles. Checkpoint kinase 1 (Chk1) is activated at replication forks that encounter damaged DNA. Subsequently, Chk1 inhibits the initiation of new replication factories and stimulates the firing of dormant origins (those in the vicinity of stalled forks). Chk1 also avoids fork collapse into DSBs (double strand breaks) and promotes fork elongation. At the molecular level, the current model considers stalled forks as the site of Chk1 activation and the nucleoplasm as the location where Chk1 phosphorylates target proteins. This model certainly serves to explain how Chk1 modulates origin firing, but how Chk1 controls the fate of stalled forks is less clear. Interestingly, recent reports demonstrating that Chk1 phosphorylates chromatin-bound proteins and even holds kinase-independent functions might shed light on how Chk1 contributes to the elongation of damaged DNA. Indeed, such findings have unveiled a puzzling connection between Chk1 and DNA lesion bypass, which might be central to promoting fork elongation and checkpoint attenuation. In summary, Chk1 is a multifaceted and versatile signaling factor that acts at ongoing forks and replication origins to determine the extent and quality of the cellular response to replication stress.

Phosphorylation and stabilization of Arabidopsis MAP kinase phosphatase 1 in response to UV-B stress.

MAP kinase phosphatases (MKPs) are important regulators of the activation levels and kinetics of MAP kinases. This is crucial for a large number of physiological processes during development and growth, as well as interactions with the environment, including the response to ultraviolet-B (UV-B) stress. Arabidopsis MKP1 is a key regulator of MAP kinases MPK3 and MPK6 in response to UV-B stress. However, virtually nothing is presently known about the post-translational regulation of plant MKPs in vivo. Here, we provide evidence that MKP1 is a phosphoprotein in vivo and that MKP1 accumulates in response to UV-B stress. Moreover, proteasome inhibitor experiments suggest that MKP1 is constantly turned-over under non-stress conditions and that MKP1 is stabilized upon stress treatment. Stress-responsive phosphorylation and stabilization of MKP1 demonstrate the post-translational regulation of a plant MKP in vivo, adding an additional regulatory layer to MAP kinase signaling in plants.

ATR and MKP1 play distinct roles in response to UV-B stress in Arabidopsis.

Ultraviolet-B (UV-B) stress activates MAP kinases (MAPKs) MPK3 and MPK6 in Arabidopsis. MAPK activity must be tightly controlled in order to ensure an appropriate cellular outcome. MAPK phosphatases (MKPs) effectively control MAPKs by dephosphorylation of phosphothreonine and phosphotyrosine in their activation loops. Arabidopsis MKP1 is an important regulator of MPK3 and MPK6, and mkp1 knockout mutants are hypersensitive to UV-B stress, which is associated with reduced inactivation of MPK3 and MPK6. Here, we demonstrate that MPK3 and MPK6 are hyperactivated in response to UV-B in plants that are deficient in photorepair, suggesting that UV-damaged DNA is a trigger of MAPK signaling. This is not due to a block in replication, as, in contrast to atr, the mkp1 mutant is not hypersensitive to the replication-inhibiting drug hydroxyurea, hydroxyurea does not activate MPK3 and MPK6, and atr is not impaired in MPK3 and MPK6 activation in response to UV-B. We further show that mkp1 leaves and roots are UV-B hypersensitive, whereas atr is mainly affected at the root level. Tolerance to UV-B stress has been previously associated with stem cell removal and CYCB1;1 accumulation. Although UV-B-induced stem cell death and CYCB1;1 expression are not altered in mkp1 roots, CYCB1;1 expression is reduced in mkp1 leaves. We conclude that the MKP1 and ATR pathways operate in parallel, with primary roles for ATR in roots and MKP1 in leaves.

Arabidopsis MAP kinase phosphatase 1 and its target MAP kinases 3 and 6 antagonistically determine UV-B stress tolerance, independent of the UVR8 photoreceptor pathway.

Plants perceive UV-B radiation as an informational signal by a pathway involving UVR8 as UV-B photoreceptor, activating photomorphogenic and acclimation responses. In contrast, the response to UV-B as an environmental stress involves mitogen-activated protein kinase (MAPK) signalling cascades. Whereas the perception pathway is plant specific, the UV-B stress pathway is more broadly conserved. Knowledge of the UV-B stress-activated MAPK signalling pathway in plants is limited, and its potential interplay with the UVR8-mediated pathway has not been defined. Here, we show that loss of MAP kinase phosphatase 1 in the mutant mkp1 results in hypersensitivity to acute UV-B stress, but without impairing UV-B acclimation. The MKP1-interacting proteins MPK3 and MPK6 are activated by UV-B stress and are hyperactivated in mkp1. Moreover, mutants mpk3 and mpk6 exhibit elevated UV-B tolerance and partially suppress the UV-B hypersensitivity of mkp1. We show further that the MKP1-regulated stress-response MAPK pathway is independent of the UVR8 photoreceptor, but that MKP1 also contributes to survival under simulated sunlight. We conclude that, whereas UVR8-mediated acclimation in plants promotes UV-B-induced defence measures, MKP1-regulated stress signalling results when UV-B protection and repair are insufficient and damage occurs. The combined activity of these two mechanisms is crucial to UV-B tolerance in plants.

Arabidopsis MAP Kinase Phosphatase 1 (AtMKP1) negatively regulates MPK6-mediated PAMP responses and resistance against bacteria.

A primary component of plant defense is the detection of pathogen-associated molecular patterns (PAMPs) by plasma membrane-localized pathogen recognition receptors. PAMP perception results in rapid and transient activation of phosphorylation-dependent signaling pathways that lead to a wide array of defense-related responses, including extensive changes in gene expression. In Arabidopsis, several kinases, including the mitogen-activated protein kinases (MAPKs) MPK6 and MPK3, are rapidly activated after PAMP treatment, and are thought to positively regulate a wide array of defense-related responses. In contrast, negative regulation of PAMP responses by downstream phosphatases remains poorly understood. Here we report the identification of Arabidopsis MAP Kinase Phosphatase 1 (MKP1) as a negative regulator of diverse PAMP responses, including activation of MPK6 and MPK3, transient production of extracellular reactive oxygen species, accumulation of a subset of PAMP-regulated transcripts, and inhibition of seedling growth. In agreement with the enhanced PAMP response phenotypes observed in the mkp1 mutant, we found that mkp1 seedlings and adult plants are more resistant to the virulent bacterial pathogen Pseudomonas syringae pv. tomato (Pto) DC3000. Further genetic analysis revealed that MPK6, but not MPK3, is required for the mkp1-dependent increase in resistance to Pto and enhanced PAMP-induced growth inhibition observed in mkp1 seedlings. Together, our data support a role for MKP1 as a negative regulator of MPK6-mediated PAMP responses.

Emerging functions for plant MAP kinase phosphatases.

Reversible phosphorylation is a crucial regulatory mechanism that controls the activity of proteins. In mitogen-activated protein kinase (MAPK) signaling cascades, the cellular response depends on the intensity and duration of the MAPK activation, which is determined by balanced phosphorylation-dephosphorylation. MAPK phosphatases (MKPs), a subgroup of the dual-specificity phosphatases, are major negative regulators of MAPKs. The plant MKP family members are highly diverse in their structure and biological functions, and can be classified into five groups by sequence analysis. We review the recent progress made by genetic studies in identifying the physiological role of plant MKPs in a multitude of cellular processes, including cytoskeleton rearrangement, stress responses and phytohormone signaling, and examine the importance of negative regulators in plant MAPK signaling networks.

MAP kinase phosphatase1 and protein tyrosine phosphatase1 are repressors of salicylic acid synthesis and SNC1-mediated responses in Arabidopsis.

Mitogen-activated protein (MAP) kinase phosphatases are important negative regulators of the levels and kinetics of MAP kinase activation that modulate cellular responses. The dual-specificity phosphatase MAP KINASE PHOSPHATASE1 (MKP1) was previously shown to regulate MAP KINASE6 (MPK6) activation levels and abiotic stress responses in Arabidopsis thaliana. Here, we report that the mkp1 null mutation in the Columbia (Col) accession results in growth defects and constitutive biotic defense responses, including elevated levels of salicylic acid, camalexin, PR gene expression, and resistance to the bacterial pathogen Pseudomonas syringae. PROTEIN TYROSINE PHOSPHATASE1 (PTP1) also interacts with MPK6, but the ptp1 null mutant shows no aberrant growth phenotype. However, the pronounced constitutive defense response of the mkp1 ptp1 double mutant reveals that MKP1 and PTP1 repress defense responses in a coordinated fashion. Moreover, mutations in MPK3 and MPK6 distinctly suppress mkp1 and mkp1 ptp1 phenotypes, indicating that MKP1 and PTP1 act as repressors of inappropriate MPK3/MPK6-dependent stress signaling. Finally, we provide evidence that the natural modifier of mkp1 in Col is largely the disease resistance gene homolog SUPPRESSOR OF npr1-1, CONSTITUTIVE 1 (SNC1) that is absent in the Wassilewskija accession. Our data thus indicate a major role of MKP1 and PTP1 in repressing salicylic acid biosynthesis in the autoimmune-like response caused by SNC1.