Biology subjects

Kettenbach, A. N.

Publications and source records attributed to Kettenbach, A. N..

18 recordsLinked to original sources

bioRxiv2026

The Case for Kinases:A Phosphorylation Driven Model for Circadian Temperature Compensation

Circadian rhythms, [~]24-hour biological cycles, enable organisms to anticipate rhythmic environmental cycles so they can assign proper day and night functions that align with those cycles. Circadian rhythms are defined by their ability to be reset by external cues, their capacity to continue to oscillate in the absence of those cues, and their capacity to maintain the rate of the clock across a range of ambient temperatures, a property known as temperature compensation. In the Neurospora clock, the White Collar Complex (WCC) drives expression of FRQ which nucleates a complex including FRH and CK1a that phosphorylates and thereby represses WCC activity. Work to date has suggested that kinases may be involved in temperature compensation and that in Neurospora the primary target of these is FRQ. Here we investigate the genetic relationship between two clock kinases, Casein Kinase I (ck-1a) and Casein Kinase II (cka), in their regulation of temperature compensation using novel alleles, ck-1aD135G and {Delta}cka. We find that that the clock relies on Casein Kinase I more at cold temperature, but this changes as temperature increases, and the clock relies more on Casein Kinase II at warm temperatures. Using quantitative proteomics on FRQ across temperatures, we find that the FRQ phosphorylation landscape is dependent on temperature and is altered in temperature compensation mutants. This leads to the development of a phosphorylation driven model for temperature compensation, where key temperature compensation specific domains on FRQ are phosphorylated to regulate period length in response to temperature, including by Casein Kinase I and Casein Kinase II.

genetics↗

bioRxiv2025

Endocrine therapy induces oxidative stress in ER+ breast cancer that sensitizes persister cells to ferroptosis

Despite endocrine therapy, recurrence and progression of estrogen receptor alpha (ER)-positive breast cancer remain significant clinical problems. We therefore sought to identify mechanisms underlying endocrine-tolerant persistence. Endocrine-tolerant persister ER+ breast cancer cells were oxidatively stressed during endocrine therapy. Proteomic analysis revealed upregulation of antioxidant-driving enzymes including glutathione peroxidase 4 (GPX4) in persisters. Relief of oxidative stress enhanced persister fitness. The increased oxidative state of persisters enabled lipid peroxidation and ferroptosis. Persisters had an altered lipidome with increased levels of polyunsaturated fatty acids prone to peroxidation, which was attributable in part to increased lysophosphatidylcholine acyltyransferase 3 (LPCAT3, MBOAT5) expression via loss of ER-mediated repression during endocrine therapy. Treatment with the GPX4 inhibitor RSL3 enhanced the anti-persister effects of endocrine-based therapies in xenograft-bearing mice. These findings supporting the development of therapeutic strategies to leverage the oxidative stress induced by endocrine-based therapies and drive ferroptosis as a treatment for ER+ breast cancer. Statement of SignificanceEndocrine therapy increases oxidative stress and sensitizes endocrine-tolerant persister ER+ breast cancer cells to ferroptosis, indicating that therapies targeting this metabolic dependency could help prevent disease recurrence and progression.

cancer biology↗

bioRxiv2024

An atlas of RNA-dependent proteins in cell division reveals the riboregulation of mitotic protein-protein interactions

Ribonucleoprotein complexes are dynamic assemblies of RNA with RNA-binding proteins (RBPs), which can modulate the fate of the RNA molecules from transcription to degradation. Vice versa, RNA can regulate the interactions and functions of the associated proteins. Dysregulation of RBPs is linked to diseases such as cancer and neurological disorders. RNA and RBPs are present in mitotic structures like the centrosomes and spindle microtubules, but their influence on mitotic spindle integrity remains unknown. Thus, we applied the R-DeeP strategy for the proteome-wide identification of RNA-dependent proteins and complexes to cells synchronized in mitosis versus interphase. The resulting atlas of RNA-dependent proteins in cell division can be accessed through the R-DeeP 3.0 database (R-DeeP3.dkfz.de). It revealed key mitotic factors as RNA-dependent such as AURKA, KIFC1 and TPX2 that were linked to RNA despite their lack of canonical RNA-binding domains. KIFC1 was identified as a new interaction partner and phosphorylation substrate of AURKA at S349 and T359. In addition, KIFC1 interacted with both, AURKA and TPX2, in an RNA-dependent manner. Our data suggest a riboregulation of mitotic protein-protein interactions during spindle assembly, offering new perspectives on the control of cell division processes by RNA-protein complexes. HighlightsO_LIDifferential R-DeeP screens in mitosis and interphase are provided as a resource in a user-friendly database at R-DeeP3.dkfz.de C_LIO_LIAn atlas of RNA-dependent proteins in cell division identifies a substantial number of unconventional RNA-binding proteins among mitotic factors C_LIO_LIInvestigation of protein-protein interactions reveals KIFC1 as a new AURKA and TPX2 interaction partner during spindle assembly C_LIO_LIKIFC1, AURKA and TPX2 interact with each other in an RNA-dependent manner and directly bind to RNA C_LIO_LIAURKA phosphorylates KIFC1 at residues S349 and T359 C_LI

cell biology↗

bioRxiv2024

PLK-1 regulates MEX-1 polarization in the C. elegans zygote

The one-cell C. elegans embryo undergoes an asymmetric cell division during which germline factors such as the RNA-binding proteins POS-1 and MEX-1 segregate to the posterior cytoplasm, leading to their asymmetric inheritance to the posterior germline daughter cell. Previous studies found that the RNA-binding protein MEX-5 recruits polo-like kinase PLK-1 to the anterior cytoplasm where PLK-1 inhibits the retention of its substrate POS-1, leading to POS-1 segregation to the posterior. In this study, we tested whether PLK-1 similarly regulates MEX-1 polarization. We find that both the retention of MEX-1 in the anterior and the segregation of MEX-1 to the posterior depend on PLK kinase activity and on the interaction between MEX-5 and PLK-1. Human PLK1 directly phosphorylates recombinant MEX-1 on 9 predicted PLK-1 sites in vitro, four of which were identified in previous phosphoproteomic analysis of C. elegans embryos. The introduction of alanine substitutions at these four PLK-1 phosphorylation sites (MEX-1(4A)) significantly weakened the inhibition of MEX-1 retention in the anterior, thereby weakening MEX-1 segregation to the posterior. In contrast, mutation of a predicted CDK1 phosphorylation site had no effect on MEX-1 retention or on MEX-1 segregation. MEX-1(4A) mutants are viable and fertile but display significant sterility and fecundity defects at elevated temperatures. Taken together with our previous findings, these findings suggest PLK-1 phosphorylation drives both MEX-1 and POS-1 polarization during the asymmetric division of the zygote.

developmental biology↗

bioRxiv2023

Acetylation of WCC is dispensable for the core circadianclock but differentially regulates acute light responses in Neurospora

In the Neurospora circadian system, the White Collar Complex (WCC) formed by WC-1 and WC-2 drives expression of the frequency (frq) gene whose product FRQ feedbacks to inhibit transcriptional activity of WCC. Phosphorylation of WCC has been extensively studied, but the extent and significance of other post-translational modifications (PTM) has been poorly studied. To this end, we used mass-spectrometry to study alkylation sites on WCC, resulting in discovery of nine acetylation sites. Mutagenesis analysis showed most of the acetylation events individually do not play important roles in period determination. Moreover, mutating all the lysines falling in either half of WC-1 or all the lysine residues in WC-2 to arginines did not abolish circadian rhythms. In addition, we also found nine mono-methylation sites on WC-1, but like acetylation, individual ablation of most of the mono-methylation events did not result in a significant period change. Taken together, the data here suggest that acetylation or mono-methylation on WCC is not a determinant of the pace of the circadian feedback loop. The finding is consistent with a model in which repression of WCCs circadian activity is controlled mainly by phosphorylation. Interestingly, light-induced expression of some light-responsive genes has been modulated in certain wc-1 acetylation mutants, suggesting that WC-1 acetylation events differentially regulate light responses.

biochemistry↗

bioRxiv2023

A crucial role for dynamic expression of components encoding the negative arm of the circadian clock

In the Neurospora circadian system, the White Collar Complex (WCC) drives expression of the principal circadian negative arm component frequency (frq). FRQ interacts with FRH (FRQ-interacting helicase) and CK-1 forming a stable complex that represses its own expression by inhibiting WCC. In this study, a genetic screen identified a gene, designated as brd-8, that encodes a conserved auxiliary subunit of the NuA4 histone acetylation complex. Loss of brd-8 reduces H4 acetylation and RNA polymerase (Pol) II occupancy at frq and other known circadian genes, and leads to a long circadian period, delayed phase, and defective overt circadian output at some temperatures. In addition to strongly associating with the NuA4 histone acetyltransferase complex, BRD-8 is also found complexed with the transcription elongation regulator BYE-1. Expression of brd-8, bye-1, histone hH2Az, and several NuA4 subunits is controlled by the circadian clock, indicating that the molecular clock both regulates the basic chromatin status and is regulated by changes in chromatin. Taken together, our data identify new auxiliary elements of the fungal NuA4 complex having homology to mammalian components, which along with conventional NuA4 subunits, are required for timely and dynamic frq expression and thereby a normal and persistent circadian rhythm.

biochemistry↗

bioRxiv2023

Quantitative proteomics and phosphoproteomics of PPP2R5D variants reveal deregulation of RPS6 phosphorylation through converging signaling cascades

Variants in the phosphoprotein phosphatase-2 regulatory protein-5D gene (PPP2R5D) cause the clinical phenotype of Jordans Syndrome (PPP2R5D-related disorder), which includes intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for [~]40% of reported cases. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G>A) in the PPP2R5D allele in a heterozygous manner in HEK293 cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of wild-type, E198K, and E420K cell lines and find unique and shared changes between variants and wild-type cells in kinase- and phosphatase-controlled signaling cascades. As shared signaling alterations, we observed ribosomal protein S6 (RPS6) hyperphosphorylation, indicative of increased ribosomal protein S6-kinase activity. Rapamycin treatment suppressed RPS6 phosphorylation in both, suggesting activation of mTORC1. Intriguingly, our data suggest AKT-dependent (E420K) and -independent (E198K) activation of mTORC1. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, treatment with rapamycin or a p70S6K inhibitor warrants further investigation as potential therapeutic strategies for patients.

cell biology↗

bioRxiv2023

Inducible protein degradation as a strategy to identify Phosphoprotein Phosphatase 6 substrates in RAS-mutant colorectal cancer cells

Protein phosphorylation is an essential regulatory mechanism that controls most cellular processes, including cell cycle progression, cell division, and response to extracellular stimuli, among many others, and is deregulated in many diseases. Protein phosphorylation is coordinated by the opposing activities of protein kinases and protein phosphatases. In eukaryotic cells, most serine/threonine phosphorylation sites are dephosphorylated by members of the Phosphoprotein Phosphatase (PPP) family. However, we only know for a few phosphorylation sites which specific PPP dephosphorylates them. Although natural compounds such as calyculin A and okadaic acid inhibit PPPs at low nanomolar concentrations, no selective chemical PPP inhibitors exist. Here, we demonstrate the utility of endogenous tagging of genomic loci with an auxin-inducible degron (AID) as a strategy to investigate specific PPP signaling. Using Protein Phosphatase 6 (PP6) as an example, we demonstrate how rapidly inducible protein degradation can be employed to identify dephosphorylation SITES and elucidate PP6 biology. Using genome editing, we introduce AID-tags into each allele of the PP6 catalytic subunit (PP6c) in DLD-1 cells expressing the auxin receptor Tir1. Upon rapid auxin-induced degradation of PP6c, we perform quantitative mass spectrometry-based proteomics and phosphoproteomics to identify PP6 substrates in mitosis. PP6 is an essential enzyme with conserved roles in mitosis and growth signaling. Consistently, we identify candidate PP6c-dependent phosphorylation sites on proteins implicated in coordinating the mitotic cell cycle, cytoskeleton, gene expression, and mitogen-activated protein kinase (MAPK) and Hippo signaling. Finally, we demonstrate that PP6c opposes the activation of large tumor suppressor 1 (LATS1) by dephosphorylating Threonine 35 (T35) on Mps One Binder (MOB1), thereby blocking the interaction of MOB1 and LATS1. Our analyses highlight the utility of combining genome engineering, inducible degradation, and multiplexed phosphoproteomics to investigate signaling by individual PPPs on a global level, which is currently limited by the lack of tools for specific interrogation.

cell biology↗

bioRxiv2022

Phosphoprotein Phosphatase Activity Positively Regulates Oligomeric Pyrin to Trigger Inflammasome Assembly in Response to Bacterial Effectors and Toxins that Inactivate RhoA in Macrophages

Pyrin is a pattern-recognition receptor in phagocytes that triggers capase-1 inflammasome assembly in response to bacterial toxins and effectors that inactivate RhoA. Pyrin contains oligomerization domains and is negatively regulated by phosphorylation of two residues, S205 and S241 (murine) or S208 and S242 (human), via the kinases PKN1/2, which are activated by RhoA. Familial Mediterranean Fever (FMF) is caused by phagocyte production of pyrin gain of function variants, which have a lower threshold for inflammasome assembly upon RhoA-PKN axis inhibition. Inactivation of the RhoA-PKN axis removes negative regulation but a phosphoprotein phosphatase (PPP) is needed to positively regulate pyrin. No PPP that dephosphorylates pyrin has been identified, oligomerization of murine pyrin has not been studied, and the phosphorylation status of oligomeric pyrin is unknown. We used murine macrophages and FMF patients monocytes combined with the use of bacterial agonists and chemical inhibitors, native PAGE, phospho-specific antibodies and siRNA knockdowns to determine if a PPP positively regulates oligomeric pyrin. Results with broadly-specific inhibitors indicate that PPP activity is required to dephosphorylate murine and human pyrin in wild type or FMF patients phagocytes. Findings from native PAGE show that murine pyrin forms oligomers that are phosphorylated on S205 prior to RhoA inactivation. Inhibitors cause reduced mobility of murine pyrin on native PAGE and hyperphosphorylation of S242 in human pyrin, suggesting a PPP constitutively counterbalances PKN to keep the second site hypophosphorylated. Data from siRNA knockdown experiments implicate PP2A in dephosphorylation of S205 and positive regulation of pyrin in response to RhoA inactivation. Key pointsMurine pyrin is oligomeric and phosphorylated on S205 prior to inflammasome assembly PPP activity positively regulates pyrin inflammasome assembly in mice and humans The alpha and beta subunits of PP2A dephosphorylate murine pyrin S205 in macrophages

immunology↗

bioRxiv2022

Feedback in the β-catenin destruction complex imparts bistability and cellular memory

Wnt ligands are considered classical morphogens, for which the strength of the cellular response is proportional to the concentration of the ligand. Herein, we show an emergent property of bistability arising from feedback among the Wnt destruction complex proteins that target the key transcriptional co-activator {beta}-catenin for degradation. Using biochemical reconstitution, we identified positive feedback between the scaffold protein Axin and the kinase GSK3. Theoretical modeling of this feedback between Axin and GSK3 predicted that the activity of the destruction complex exhibits bistable behavior. We experimentally confirmed these predictions by demonstrating that cellular cytoplasmic {beta}-catenin concentrations exhibit an "all-or-none" response with sustained memory (hysteresis) of the signaling input. This bistable behavior was transformed into a graded response and memory was lost through inhibition of GSK3. These findings provide a mechanism for establishing decisive, switch-like cellular response and memory upon Wnt pathway stimulation. One Sentence SummaryPositive feedback within the {beta}-catenin destruction complex gives rise to bistability and memory in response to Wnt stimulation, imparting signal transduction accuracy and insulation.

cell biology↗

bioRxiv2021

Regulation of PP2A, PP4, and PP6 holoenzyme assembly by carboxyl-terminal methylation

The family of Phosphoprotein Phosphatases (PPPs) is responsible for most cellular serine and threonine dephosphorylation. PPPs achieve substrate specificity and selectivity by forming multimeric holoenzymes. PPP holoenzyme assembly is tightly controlled, and changes in the cellular repertoire of PPPs are linked to human disease, including cancer and neurodegeneration. For PP2A, PP4, and PP6, holoenzyme formation is in part regulated by carboxyl (C)-terminal methyl-esterification (often referred to as "methylation"). Here, we use mass spectrometry-based proteomics, methylation-ablating mutations, and genome editing to elucidate the role of C-terminal methylation on PP2A, PP4, and PP6 holoenzyme assembly. We find that the catalytic subunits of PP2A, PP4, and PP6 are frequently methylated in cancer cells and that deletion of the C-terminal leucine faithfully recapitulates loss of methylation. We observe that loss of PP2A methylation consistently reduced B55, B56, and B72 regulatory subunit binding in cancer and non-transformed cell lines. However, Striatin subunit binding is only affected in non-transformed cells. For PP4, we find that PP4R1 and PP4R3{beta} bind in a methylation-dependent manner. Intriguingly, loss of methylation does not affect PP6 holoenzymes. Our analyses demonstrate in an unbiased, comprehensive, and isoform-specific manner the crucial regulatory function of endogenous PPP methylation in transformed and non-transformed cell lines

biochemistry↗

bioRxiv2021

The Candida albicans Cdk8-dependent phosphoproteome reveals repression of hyphal growth through a Flo8-dependent pathway

Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans, the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1. Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent activity, suggesting that Ssn3 kinase activity negatively regulates Flo8. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that FLO8 and STP2, a transcription factor involved in amino acid utilization, are required for ssn3{Delta}/{Delta} phenotype, but that loss of the Ssn3 phosphosites identified in Flo8 was not sufficient to phenocopy the ssn3{Delta}/{Delta} mutant. These data highlight the spectrum of processes affected by the modulation of Ssn3 activity and underscore the importance of considering Ssn3 function in the control of transcription factor activities.

microbiology↗

bioRxiv2021

PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107

Protein phosphorylation is a reversible post-translation modification essential in cell signaling. This study addresses a long-standing question as to how the most abundant serine/threonine Protein Phosphatase 2 (PP2A) holoenzyme, PP2A/B55, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55 recruits p107, a pRB-related tumor suppressor and B55 substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615-626) encompassing the strongest p107 binding site. This enabled us to identify an "HxRVxxV619-625" short linear motif (SLiM) in p107 as necessary for B55 binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55/PP2A substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, "p[ST]-P-x(5-10)-[RK]-V-x-x-[VI]-R". Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55 groove, and phosphosite presentation. Altogether these data provide key insights into PP2A/B55 mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55[<] role in multiple cellular processes.

biochemistry↗

bioRxiv2020

Selective dephosphorylation by PP2A-B55 directs the meiosis I - meiosis II transition in oocytes

Meiosis is a specialized cell cycle that requires sequential changes to the cell division machinery to facilitate changing functions. To define the mechanisms that enable the oocyte-to-embryo transition, we performed time-course proteomics in sea star oocytes from prophase I through the first embryonic cleavage. Although protein levels are broadly stable, dynamic waves of phosphorylation underlie each meiotic stage. We find that the phosphatase PP2A-B55 is reactivated at the Meiosis I/II transition resulting in the preferential dephosphorylation of threonine residues. Selective dephosphorylation is critical for directing the MI / MII transition as altering PP2A-B55 substrate preferences disrupts key cell cycle events after meiosis I. In addition, threonine to serine substitution of a conserved phosphorylation site in the substrate INCENP prevents its relocalization at anaphase I. Thus, through its inherent phospho-threonine preference, PP2A-B55 rewires the cell division apparatus to direct the MI / MII transition.

cell biology↗

bioRxiv2020

STK32A is a dual-specificity AGC kinase with a preference for acidic substrates

The STK32 kinases are a small subfamily of three uncharacterised serine/threonine kinases from the AGC kinase family whose functional role is so far unknown. Here, we analyse the consensus peptide sequence for STK32A phosphorylation, showing that STK32A is directed towards acidic substrate sequences and exhibits dual-specificity for serine/threonine and tyrosine residues. A crystal structure of STK32A reveals an overall structure typical of the AGC protein kinase family but with significant and unique features including an altered binding mode of the hydrophobic motif to the N-terminal lobe of the kinase domain, and a novel alpha-helix in between the turn motif and the hydrophobic motif. The crystal structure combined with phylogenetic analysis reveals the evolutionary conservation of the acidic substrate preference. In vitro binding assays demonstrated that the STK32 kinases bind significant numbers of clinically used kinase inhibitors.

biochemistry↗

bioRxiv2019

A mechanism for how Cdr1/Nim1 kinase promotes mitotic entry by inhibiting Wee1

To enter into mitosis, cells must shut off the cell cycle inhibitor Wee1. SAD family protein kinases regulate Wee1 signaling in yeast and humans. In S. pombe, two SAD kinases (Cdr1/Nim1 and Cdr2) act as upstream inhibitors of Wee1. Previous studies found that S. pombe Cdr1/Nim1 directly phosphorylates and inhibits Wee1 in vitro, but different results were obtained for budding yeast and human SAD kinases. Without a full understanding of Cdr1 action on Wee1, it has been difficult to assess the in vivo relevance and conservation of this mechanism. Here, we show that both Cdr1 and Cdr2 promote Wee1 phosphorylation in cells, but only Cdr1 inhibits Wee1 kinase activity. Inhibition occurs when Cdr1 phosphorylates a cluster of serine residues linking -helices G and H of the Wee1 kinase domain. This region is highly divergent among different Wee1 proteins, consistent with distinct regulatory mechanisms. A wee(4A) mutant that impairs phosphorylation by Cdr1 delays mitotic entry and causes elongated cells. By disrupting and re-targeting Cdr1 localization, we show that Cdr1 inhibition of Wee1 occurs in cells at cortical nodes formed by Cdr2. Based on our results, we propose a two-step model for inhibition of Wee1 by Cdr1 and Cdr2 at nodes.

cell biology↗

bioRxiv2019

Defining how Pak1 regulates cell polarity and cell division in fission yeast

Protein kinases direct polarized growth by regulating the cytoskeleton in time and space, and could play similar roles in cell division. We found that the Cdc42-activated polarity kinase Pak1 colocalizes with the assembling contractile actomyosin ring (CAR) and remains at the division site during septation. Mutations in pak1 led to defects in CAR assembly and genetic interactions with cytokinesis mutants. Through a phosphoproteomic screen, we identified novel Pak1 substrates that function in polarized growth and cytokinesis. For cytokinesis, we found that Pak1 regulates the localization of its substrates Mid1 and Cdc15 to the CAR. Mechanistically, Pak1 phosphorylates the Mid1 N-terminus to promote its association with cortical nodes that act as CAR precursors. Defects in Pak1-Mid1 signaling lead to misplaced and defective division planes, but these phenotypes can be rescued by synthetic tethering of Mid1 to cortical nodes. Our work defines a new signaling mechanism driven by a cell polarity kinase that promotes CAR assembly in the correct time and place. SummaryMagliozzi et al. show that fission yeast cell polarity kinase Pak1 regulates cytokinesis. Through a phosphoproteomic screen and subsequent mutant analysis, their work uncovers direct targets and mechanisms for Pak1 activity during cell division.

cell biology↗

bioRxiv2018

A complex containing lysine-acetylated actin inhibits the formin INF2

INF2 is a member of the formin family of actin assembly factors. Dominant mis-sense mutations in INF2 link to two diseases: focal segmental glomerulosclerosis (FSGS), a kidney disease; and Charcot-Marie-Tooth disease (CMTD), a neuropathy. All disease mutations map to the autoinhibitory Diaphanous Inhibitory Domain (DID). Curiously, purified INF2 is not autoinhibited, suggesting the existence of additional cellular inhibitors. We purified an INF2 inhibitor from mouse brain, and identified it as a complex between lysine-acetylated actin (KAc-actin) and cyclase-associated protein (CAP). Inhibition of INF2 by CAP/KAc-actin requires INF2 DID. Treatment of CAP/KAc-actin with histone deacetylase 6 (HDAC6) releases INF2 inhibition, while HDAC6 inhibitors block cellular INF2 activation. INF2 disease mutants are poorly inhibited by CAP/KAc-actin, suggesting that FSGS and CMTD result from reduced CAP/KAc-actin binding. This is the first demonstrated role for lysine-acetylated actin: regulation of an actin assembly factor by a novel mechanism, which we call facilitated auto-inhibition.

biochemistry↗