The HIPK2/CDC14B-MeCP2 axis enhances the spindle assembly checkpoint block by promoting cyclin B translation.

Mitotic perturbations activate the spindle assembly checkpoint (SAC) that keeps cells in prometaphase with high CDK1 activity. Prolonged mitotic arrest is eventually bypassed by gradual cyclin B decline followed by slippage of cells into G1 without chromosome segregation, a process that promotes cell transformation and drug resistance. Hitherto, the cyclin B1 decay is exclusively defined by mechanisms that involve its proteasomal degradation. Here, we report that hyperphosphorylated HIPK2 kinase accumulates in mitotic cells and phosphorylates the Rett syndrome protein MeCP2 at Ser92, a regulation that is counteracted by CDC14B phosphatase. MeCP2S92 phosphorylation leads to the enhanced translation of cyclin B1, which is important for cells with persistent SAC activation to counteract the proteolytic decline of cyclin B1 and therefore to suspend mitotic slippage. Hence, the HIPK2/CDC14B-MeCP2 axis functions as an enhancer of the SAC-induced mitotic block. Collectively, our study revises the prevailing view of how cells confer a sustainable SAC.

PEAK1 Y635 phosphorylation regulates cell migration through association with Tensin3 and integrins.

Integrins mediate cell adhesion by connecting the extracellular matrix to the intracellular cytoskeleton and orchestrate signal transduction in response to chemical and mechanical stimuli by interacting with many cytoplasmic proteins. We used BioID to interrogate the interactomes of ß1 and ß3 integrins in epithelial cells and identified PEAK1 as an interactor of the RGD-binding integrins α5ß1, αVß3, and αVß5 in focal adhesions. We demonstrate that the interaction between integrins and PEAK1 occurs indirectly through Tensin3, requiring both the membrane-proximal NPxY motif on the integrin ß tail and binding of the SH2 domain of Tensin3 to phosphorylated Tyr-635 on PEAK1. Phosphorylation of Tyr-635 is mediated by Src and regulates cell migration. Additionally, we found that Shc1 localizes in focal adhesions in a PEAK1 phosphorylated Tyr-1188-dependent fashion. Besides binding Shc1, PEAK1 also associates with a protein cluster that mediates late EGFR/Shc1 signaling. We propose a model in which PEAK1 binds Tensin3 and Shc1 to converge integrin and growth factor receptor signal transduction.

CDK1-cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry.

The disassembly of integrin-containing focal adhesions (FAs) at mitotic entry is essential for cell rounding, mitotic retraction fibre formation, bipolar spindle positioning and chromosome segregation. The mechanism that drives FA disassembly at mitotic entry is unknown. Here, we show that the CDK1-cyclin B1 complex phosphorylates the integrin activator kindlin, which results in the recruitment of the cullin 9-FBXL10 ubiquitin ligase complex that mediates kindlin ubiquitination and degradation. This molecular pathway is essential for FA disassembly and cell rounding, as phospho-inhibitory mutations of the CDK1 motif prevent kindlin degradation, FA disassembly and mitotic cell rounding. Conversely, phospho-mimetic mutations promote kindlin degradation in interphase, accelerate mitotic cell rounding and impair mitotic retraction fibre formation. Despite the opposing effects on kindlin stability, both types of mutations cause severe mitotic spindle defects, apoptosis and aneuploidy. Thus, the exquisite regulation of kindlin levels at mitotic entry is essential for cells to progress accurately through mitosis.

The human phosphatase CDC14A modulates primary cilium length by regulating centrosomal actin nucleation.

CDC14A codes for a conserved proline-directed phosphatase, and mutations in the gene are associated with autosomal-recessive severe to profound deafness, due to defective kinocilia. A role of CDC14A in cilia formation has also been described in other organisms. However, how human CDC14A impacts on cilia formation remains unclear. Here, we show that human RPE1 hCDC14APD cells, encoding a phosphatase dead version of hCDC14A, have longer cilia than wild-type cells, while hCDC14A overexpression reduces cilia formation. Phospho-proteome analysis of ciliated RPE1 cells identified actin-associated and microtubule binding proteins regulating cilia length as hCDC14A substrates, including the actin-binding protein drebrin. Indeed, we find that hCDC14A counteracts the CDK5-dependent phosphorylation of drebrin at S142 during ciliogenesis. Further, we show that drebrin and hCDC14A regulate the recruitment of the actin organizer Arp2 to centrosomes. In addition, during ciliogenesis hCDC14A also regulates endocytosis and targeting of myosin Va vesicles to the basal body in a drebrin-independent manner, indicating that it impacts primary cilia formation in a multilayered manner.

The Kank family proteins in adhesion dynamics.

Integrin-mediated cell adhesion plays key roles for cell movement during development and tissue homeostasis. The dynamic life cycle of various integrin adhesions structures is required for the cell movements and regulated by the coordinated actions of both actomyosin and the microtubule (MT) cytoskeleton. The evolutionarily conserved Kank family proteins have emerged as regulators of adhesion dynamics by coordinating integrin-mediated force transmission with the recruitment of microtubules to integrins. These novel functions may play important roles in vivo and in human diseases.

Human phosphatase CDC14A regulates actin organization through dephosphorylation of epithelial protein lost in neoplasm.

CDC14 is an essential dual-specificity phosphatase that counteracts CDK1 activity during anaphase to promote mitotic exit in Saccharomyces cerevisiae Surprisingly, human CDC14A is not essential for cell cycle progression. Instead, it regulates cell migration and cell adhesion. Little is known about the substrates of hCDC14A and the counteracting kinases. Here, we combine phospho-proteome profiling and proximity-dependent biotin identification to identify hCDC14A substrates. Among these targets were actin regulators, including the tumor suppressor eplin. hCDC14A counteracts EGF-induced rearrangements of actin cytoskeleton by dephosphorylating eplin at two known extracellular signal-regulated kinase sites, serine 362 and 604. hCDC14APD and eplin knockout cell lines exhibited down-regulation of E-cadherin and a reduction in α/ß-catenin at cell-cell adhesions. Reduction in the levels of hCDC14A and eplin mRNA is frequently associated with colorectal carcinoma and is correlated with poor prognosis. We therefore propose that eplin dephosphorylation by hCDC14A reduces actin dynamics to restrict tumor malignancy.

Human phosphatase CDC14A is recruited to the cell leading edge to regulate cell migration and adhesion.

Cell adhesion and migration are highly dynamic biological processes that play important roles in organ development and cancer metastasis. Their tight regulation by small GTPases and protein phosphorylation make interrogation of these key processes of great importance. We now show that the conserved dual-specificity phosphatase human cell-division cycle 14A (hCDC14A) associates with the actin cytoskeleton of human cells. To understand hCDC14A function at this location, we manipulated native loci to ablate hCDC14A phosphatase activity (hCDC14A(PD)) in untransformed hTERT-RPE1 and colorectal cancer (HCT116) cell lines and expressed the phosphatase in HeLa FRT T-Rex cells. Ectopic expression of hCDC14A induced stress fiber formation, whereas stress fibers were diminished in hCDC14A(PD) cells. hCDC14A(PD) cells displayed faster cell migration and less adhesion than wild-type controls. hCDC14A colocalized with the hCDC14A substrate kidney- and brain-expressed protein (KIBRA) at the cell leading edge and overexpression of KIBRA was able to reverse the phenotypes of hCDC14A(PD) cells. Finally, we show that ablation of hCDC14A activity increased the aggressive nature of cells in an in vitro tumor formation assay. Consistently, hCDC14A is down-regulated in many tumor tissues and reduced hCDC14A expression is correlated with poorer survival of patients with cancer, to suggest that hCDC14A may directly contribute to the metastatic potential of tumors. Thus, we have uncovered an unanticipated role for hCDC14A in cell migration and adhesion that is clearly distinct from the mitotic and cytokinesis functions of Cdc14/Flp1 in budding and fission yeast.

Genome editing through large insertion leads to the skipping of targeted exon.

BACKGROUND: Highly efficient genome editing can be achieved through targeting an endonuclease to specific locus of interest. Engineered zinc-finger nuclease (ZFN) and CRISPR-associated protein-9 nuclease (Cas9) offer such an elegant approach for genome editing in vertebrate cells. In this study, we have utilized ZFN and Cas9-catalyzed double strand break followed by homologous recombination-mediated incorporation of premature stop codon and selection marker to target human cell division cycle 14A (hCDC14A) and cell division cycle 14B (hCDC14B) genes. RESULTS: Targeting of the hCDC14A and hCDC14B loci in telomerase immortalized human retinal pigment epithelium (hTERT-RPE1) and human colon cancer (HCT116) cells were confirmed by Southern blot hybridization. Nevertheless, DNA sequence analysis of reverse transcription polymerase chain reaction (RT-PCR) products confirmed that in all the single/double allele ablations, the targeted exon was spliced out. The phenomenon of exon skipping was independent of the genome editing approaches exploited, Cas9 or ZFN. Because the exons had a nucleotide number that could be divided by 3, the reading frame of the exon deletion was maintained. This indicates an exon-skipping event possibly due to the insertion of large DNA fragment (1.7 to 2.5 Kb) within the targeted exons. As a proof-of-principle, we have used gene disruption followed by non-homologous end joining (NHEJ) approach. Small alterations in the exon (one to fifteen bases) were transcribed to mRNA without exon skipping. Furthermore, loxP site-mediated removal of selection markers left a 45 bp scar within the targeted exon that can be traced in mRNA without exon skipping. CONCLUSION: From this study, we conclude that insertion of a large DNA fragment into an exon by genome editing can lead to its skipping from the final transcript. Hence, more cautious approach needs to be taken while designing target sites in such that the possible skipping of targeted exon causes a frame-shift mediated incorporation of pre-mature stop codon. On the other hand, exon skipping may be a useful strategy for the introduction of protein deletions.

Global phosphoproteomic effects of natural tyrosine kinase inhibitor, genistein, on signaling pathways.

Genistein is a natural protein tyrosine kinase inhibitor that exerts anti-cancer effect by inducing G2/M arrest and apoptosis. However, the phosphotyrosine signaling pathways mediated by genistein are largely unknown. In this study, we combined tyrosine phosphoprotein enrichment with MS-based quantitative proteomics technology to globally identify genistein-regulated tyrosine phosphoproteins aiming to depict genistein-inhibited phosphotyrosine cascades. Our experiments resulted in the identification of 213 phosphotyrosine sites on 181 genistein-regulated proteins. Many identified phosphoproteins, including nine protein kinases, eight receptors, five protein phosphatases, seven transcriptical regulators and four signal adaptors, were novel inhibitory effectors with no previously known function in the anti-cancer mechanism of genistein. Functional analysis suggested that genistein-regulated protein tyrosine phosphorylation mainly by inhibiting the activity of tyrosine kinase EGFR, PDGFR, insulin receptor, Abl, Fgr, Itk, Fyn and Src. Core signaling molecules inhibited by genistein can be functionally categorized into the canonial Receptor-MAPK or Receptor-PI3K/AKT cascades. The method used here may be suitable for the identification of inhibitory effectors and tyrosine kinases regulated by anti-cancer drugs.