ß-catenin turnover is regulated by Nek10-mediated tyrosine phosphorylation in A549 lung adenocarcinoma cells.

ß-catenin has influential roles affecting embryonic development, tissue homeostasis, and human diseases including cancer. Cellular ß-catenin levels are exquisitely controlled by a variety of regulatory mechanisms. In the course of exploring the functions of the Nek10 tyrosine kinase, we observed that deletion of Nek10 in lung adenocarcinoma cells resulted in dramatic stabilization of ß-catenin, suggestive of a Nek10 role in the control of ß-catenin turnover. Nek10-deficient cells exhibited diminished ability to form tumorspheres in suspension, grow in soft agar, and colonize mouse lung tissue following tail vein injection. Mechanistically, Nek10 associates with the Axin complex, responsible for ß-catenin degradation, where it phosphorylates ß-catenin at Tyr30, located within the regulatory region governing ß-catenin turnover. In the absence of Nek10 phosphorylation, GSK3-mediated phosphorylation of ß-catenin, a prerequisite for its turnover, is impaired. This represents a divergent function within the Nek family, whose other members are serine-threonine kinases involved in different elements of the centrosomal cycle, primary cilia function, and DNA damage responses.

NEK10 tyrosine phosphorylates p53 and controls its transcriptional activity.

In response to genotoxic stress, multiple kinase signaling cascades are activated, many of them directed towards the tumor suppressor p53, which coordinates the DNA damage response (DDR). Defects in DDR pathways lead to an accumulation of mutations that can promote tumorigenesis. Emerging evidence implicates multiple members of the NimA-related kinase (NEK) family (NEK1, NEK10, and NEK11) in the DDR. Here, we describe a function for NEK10 in the regulation of p53 transcriptional activity through tyrosine phosphorylation. NEK10 loss increases cellular proliferation by modulating the p53-dependent transcriptional output. NEK10 directly phosphorylates p53 on Y327, revealing NEK10's unexpected substrate specificity. A p53 mutant at this site (Y327F) acts as a hypomorph, causing an attenuated p53-mediated transcriptional response. Consistently, NEK10-deficient cells display heightened sensitivity to DNA-damaging agents. Further, a combinatorial score of NEK10 and TP53-target gene expression is an independent predictor of a favorable outcome in breast cancers.

The Rho-guanine nucleotide exchange factor PDZ-RhoGEF governs susceptibility to diet-induced obesity and type 2 diabetes.

Adipose tissue is crucial for the maintenance of energy and metabolic homeostasis and its deregulation can lead to obesity and type II diabetes (T2D). Using gene disruption in the mouse, we discovered a function for a RhoA-specific guanine nucleotide exchange factor PDZ-RhoGEF (Arhgef11) in white adipose tissue biology. While PDZ-RhoGEF was dispensable for a number of RhoA signaling-mediated processes in mouse embryonic fibroblasts, including stress fiber formation and cell migration, it's deletion led to a reduction in their proliferative potential. On a whole organism level, PDZ-RhoGEF deletion resulted in an acute increase in energy expenditure, selectively impaired early adipose tissue development and decreased adiposity in adults. PDZ-RhoGEF-deficient mice were protected from diet-induced obesity and T2D. Mechanistically, PDZ-RhoGEF enhanced insulin/IGF-1 signaling in adipose tissue by controlling ROCK-dependent phosphorylation of the insulin receptor substrate-1 (IRS-1). Our results demonstrate that PDZ-RhoGEF acts as a key determinant of mammalian metabolism and obesity-associated pathologies.

Nek family of kinases in cell cycle, checkpoint control and cancer.

Early studies in lower Eukaryotes have defined a role for the members of the NimA related kinase (Nek) family of protein kinases in cell cycle control. Expansion of the Nek family throughout evolution has been accompanied by their broader involvement in checkpoint regulation and cilia biology. Moreover, mutations of Nek family members have been identified as drivers behind the development of ciliopathies and cancer. Recent advances in studying the physiological roles of Nek family members utilizing mouse genetics and RNAi-mediated knockdown are revealing intricate associations of Nek family members with fundamental biological processes. Here, we aim to provide a comprehensive account of our understanding of Nek kinase biology and their involvement in cell cycle, checkpoint control and cancer.

m-Calpain is required for preimplantation embryonic development in mice.

BACKGROUND: Mu-calpain and m-calpain are ubiquitously expressed proteases implicated in cellular migration, cell cycle progression, degenerative processes and cell death. These heterodimeric enzymes are composed of distinct catalytic subunits, encoded by Capn1 (mu-calpain) or Capn2 (m-calpain), and a common regulatory subunit encoded by Capn4. Disruption of the mouse Capn4 gene abolished both mu-calpain and m-calpain activity, and resulted in embryonic lethality, thereby suggesting essential roles for one or both of these enzymes during mammalian embryogenesis. Disruption of the Capn1 gene produced viable, fertile mice implying that either m-calpain could compensate for the loss of mu-calpain, or that the loss of m-calpain was responsible for death of Capn4-/- mice. RESULTS: To distinguish between the alternatives described above, we deleted an essential coding region in the mouse Capn2 gene in embryonic stems cells and transmitted this mutant allele through the mouse germline. Breeding of heterozygous animals failed to produce homozygous mutant live offspring or implanted embryos. A nested PCR genotyping protocol was established, and homozygous preimplantation mutant embryos were detected at the morula but not at the blastocyts stage. CONCLUSION: We conclude that homozygous disruption of the Capn2 gene results in pre-implantation embryonic lethality between the morula and blastocyst stage. This establishes that mu-calpain and m-calpain have distinct functions, and that m-calpain is vital for development of the preimplantation murine embryo.

Expression of human, mouse, and rat m-calpains in Escherichia coli and in murine fibroblasts.

The two best known calpains, micro- and m-calpain, are Ca(2+)-dependent cysteine proteases found in all mammalian tissues. They are probably involved in many Ca(2+)-linked signal pathways, although the details are not yet clear. The enzymes are heterodimers of a specific large subunit (micro-80k or m-80k) and a common small subunit (28k). Recombinant calpains have been obtained by co-expression of large and small subunits in Escherichia coli and in Sf9 cells, with variable success. Expression with the 28k subunit is very low, but is much higher with a C-terminal 21k fragment of this subunit. Rat m-calpain (m-80k/21k) is well expressed in E. coli but mouse m-calpain (m-80k/21k) is poorly expressed, even though the amino acid sequences of rat-m-80k and mouse-m-80k are 92% identical. It had also been reported that human m-calpain could be expressed in Sf9 cells but not in E. coli. To investigate these differences, hybrid rat/mouse and rat/human m-calpains were cloned and expressed in E. coli. It was shown that Ile-6 and Pro-127, which are specific to the mouse m-80k sequence, caused poor expression. High expression of human m-calpain in E. coli could be achieved by providing the correct Shine-Dalgarno ribosome binding site. The results provide a simple method to obtain approximately 10mg amounts of human m-calpain and a slightly modified mouse m-calpain. Expression of m-80k-EGFP fusions was also studied, both in E. coli and in mammalian cells, varying both the small subunit and the promoters. m-80k-EGFP alone was not active, but with 21k or 28k subunits was active in both cell types. The EGFP domain was partially cleaved during expression, releasing an active m-80k/21k calpain.

Calpain is required for MMP-2 and u-PA expression in SV40 large T-antigen-immortalized cells.

The absence of both mu- and m-calpain activity, caused by disruption of the capn4 gene in mice, retarded migration, and disrupted the cytoskeleton, both in primary capn4(-/-) embryonic fibroblasts (mEF) and in capn4(-/-) mEF immortalized with SV40 large T-antigen (TAg). These results are thought to reflect the role of calpain in integrin signaling to the cytoskeleton. The integrins are also involved, together with matrix metalloproteinases (MMP) and plasminogen activators (PA), in cellular invasion. This study therefore aimed to establish whether links exist between the calpain, MMP, and PA systems, using both primary and TAg-immortalized capn4(+/+) and capn4(-/-) embryonic fibroblasts. Both Matrigel invasion, and expression of MMP-2 and u-PA activities, correlated with calpain expression in TAg-containing cells, but not in primary cells. MMP-2 mRNA synthesis also correlated with calpain expression in the presence of TAg, but u-PA mRNA synthesis was not so correlated. The results suggest that calpain acquires new regulatory roles in the presence of TAg. Calpain is also required for v-Src-mediated transformation. It appears that calpain may have previously unsuspected roles in oncogenic transformation.

Origins of the difference in Ca2+ requirement for activation of mu- and m-calpain.

The mu- and m-calpains are closely related Ca(2+)-dependent cysteine proteases having different in vitro Ca(2+) requirements ( K (d)), of approx. 25 and 325 microM respectively. The two isoforms are heterodimers of slightly different large (80 kDa) subunits and an identical small (28 kDa) subunit, so that the difference in K (d) values must reside in the large subunits. As assayed here, these K (d) values relate to the Ca(2+) required for the first phase of calpain activation and do not reflect the lower Ca(2+) then required by fully activated calpain. On the basis of sequence comparison and the X-ray structure of m-calpain, many m-type residues in the C-terminal EF-hand-containing domain IV were converted into the corresponding mu-type residues, but these mutations did not produce the expected decrease in K (d). In a series of hybrid (mu/m) large-subunit calpains, the K (d) values decreased progressively towards that of mu-calpain as the proportion of mu-type sequence increased from 0 to 90%. K (d) values cannot therefore be ascribed to one or a few specific intramolecular interactions, but reflect the global response of the whole molecule to Ca(2+) binding. Nonetheless, 25% of the difference in K (d) values between mu- and m-calpain can be ascribed to the N-terminal peptide of the large subunit, whereas the C-terminal EF-hand-containing domain IV accounts for 65% of the difference.