The NDR/LATS protein kinases in neurobiology: Key regulators of cell proliferation, differentiation and migration in the ocular and central nervous system.

Nuclear Dbf2-related (NDR) kinases are a subgroup of evolutionarily conserved AGC protein kinases that regulate various aspects of cell growth and morphogenesis. There are 4 NDR protein kinases in mammals, LATS1, LATS2 and STTK8/NDR1, STK38L/NDR2 protein kinases. LATS1 and 2 are core components of the well-studied Hippo pathway, which play a critical role in the regulation of cell proliferation, differentiation, and cell migration via YAP/TAZ transcription factor. The Hippo pathways play an important role in nervous tissue development and homeostasis, especially with regard to the central nervous system (CNS) and the ocular system. The ocular system is a very complex system generated by the interaction in a very tightly coordinated manner of numerous and diverse developing tissues, such as, but not limited to choroidal and retinal blood vessels, the retinal pigmented epithelium and the retina, a highly polarized neuronal tissue. The retina development and maintenance require precise and coordinated regulation of cell proliferation, cell death, migration, morphogenesis, synaptic connectivity, and balanced homeostasis. This review highlights the emerging roles of NDR1 and NDR2 kinases in the regulation of retinal/neuronal function and homeostasis via a noncanonical branch of the Hippo pathway. We highlight a potential role of NDR1 and NDR2 kinases in regulating neuronal inflammation and as potential therapeutic targets for the treatment of neuronal diseases.

Ndr kinases regulate retinal interneuron proliferation and homeostasis.

Ndr2/Stk38l encodes a protein kinase associated with the Hippo tumor suppressor pathway and is mutated in a naturally-occurring canine early retinal degeneration (erd). To elucidate the retinal functions of Ndr2 and its paralog Ndr1/Stk38, we generated Ndr1 and Ndr2 single knockout mice. Although retinal lamination appeared normal in these mice, Ndr deletion caused a subset of Pax6-positive amacrine cells to proliferate in differentiated retinas, while concurrently decreasing the number of GABAergic, HuD and Pax6-positive amacrine cells. Retinal transcriptome analyses revealed that Ndr2 deletion increased expression of neuronal stress genes and decreased expression of synaptic organization genes. Consistent with the latter, Ndr deletion dramatically reduced levels of Aak1, an Ndr substrate that regulates vesicle trafficking. Our findings indicate that Ndr kinases are important regulators of amacrine and photoreceptor cells and suggest that Ndr kinases inhibit the proliferation of a subset of terminally differentiated cells and modulate interneuron synapse function via Aak1.

Cbk1 kinase and Bck2 control MAP kinase activation and inactivation during heat shock.

Saccharomyces cerevisiae Cbk1 kinase is a LATS/NDR tumor suppressor orthologue and component of the Regulation of Ace2 and Morphogenesis signaling network. Cbk1 was previously implicated in regulating polarized morphogenesis, gene expression, and cell integrity. Here we establish that Cbk1 is critical for heat shock and cell wall stress signaling via Bck2, a protein associated with the Pkc1-Mpk1 cell integrity pathway. We demonstrate that cbk1 and bck2 loss-of-function mutations prevent Mpk1 kinase activation and Mpk1-dependent gene expression but do not disrupt Mpk1 Thr-190/Tyr-192 phosphorylation. Bck2 overexpression partially restores Mpk1-dependent Rlm1 transcription factor activity in cbk1 mutants, suggesting that Bck2 functions downstream of Cbk1. We demonstrate that Bck2 precisely colocalizes with the mitogen-activated protein kinase (MAPK) phosphatase Sdp1. During heat shock, Bck2 and Sdp1 transiently redistribute from nuclei and the cytosol to mitochondria and other cytoplasmic puncta before returning to their pre-stressed localization patterns. Significantly, Cbk1 inhibition delays the return of Bck2 and Sdp1 to their pre-stressed localization patterns and delays Mpk1 Thr-190/Tyr-192 dephosphorylation upon heat shock adaptation. We conclude that Cbk1 and Bck2 are required for Mpk1 activation during heat shock and cell wall stress and for Mpk1 dephosphorylation during heat shock adaptation. These data provide the first evidence that Cbk1 kinase regulates MAPK-dependent stress signaling and provide mechanistic insight into Sdp1 phosphatase regulation.

Nucleocytoplasmic shuttling of Ssd1 defines the destiny of its bound mRNAs.

Mechanisms that control mRNA metabolism are critical for cell function, development and stress response. The Saccharomyces cerevisiae mRNA-binding protein Ssd1 has been implicated in mRNA processing, ageing, stress response and maintenance of cell integrity. Ssd1 is a substrate of the LATS/NDR tumour suppressor orthologue Cbk1 kinase. Previous data indicate that Ssd1 localizes to the cytoplasm; however, biochemical interactions suggest that Ssd1 at least transiently localizes to the nucleus. We therefore explored whether nuclear localization is important for Ssd1 cytoplasmic functions. We identified a functional NLS in the N-terminal domain of Ssd1. An Ssd1-derived NLS-GFP fusion protein and several C-terminally truncated Ssd1 proteins, which presumably lack nuclear export sequences, accumulate in the nucleus. Alanine substitution of the Ssd1 NLS prevents Ssd1 nuclear entry, mRNA binding and disrupts Srl1 mRNA localization. Moreover, Ssd1-NLS mutations abolish Ssd1 toxicity in the absence of Cbk1 phosphorylation and cause Ssd1 to localize prominently to cytoplasmic puncta. These data indicate that nuclear shuttling is critical for Ssd1 mRNA binding and Ssd1-mRNA localization in the cytoplasm. Collectively these data support the model that Ssd1 functions analogously to hnRNPs, which bind mRNA co-transcriptionally, are exported to the cytoplasm and target mRNAs to sites of localized translation and P-bodies.

Defective in mitotic arrest 1/ring finger 8 is a checkpoint protein that antagonizes the human mitotic exit network.

A molecular pathway homologous to the S. cerevisiae mitotic exit network (MEN) and S. pombe septation initiation network has recently been described in higher eukaryotes and involves the tumor suppressor kinase LATS1 and its subunit MOB1A. The yeast MEN/septation initiation network pathways are regulated by the ubiquitin ligase defective in mitotic arrest 1 (Dma1p), a checkpoint protein that helps maintain prometaphase arrest when cells are exposed to microtubule poisons. We identified here the RING domain protein ring finger 8 (RNF8) as the human orthologue of the yeast protein Dma1p. Like its yeast counterparts, human DMA1/RNF8 localized at the midbody and its depletion by siRNA compromised mitotic arrest of nocodazole-treated cells in a manner dependent on the MEN. Depletion of MAD2, a spindle checkpoint protein, also compromised mitotic arrest, but in a MEN-independent manner. Thus, two distinct checkpoint pathways maintain mitotic arrest in cells exposed to microtubule poisons.

Human LATS1 is a mitotic exit network kinase.

The kinase LATS/WARTS is a tumor suppressor protein conserved in evolution, but its function at the molecular level is not well understood. We report here that human LATS1 interacts with MOB1A, a protein whose homologue in budding yeast associates with kinases involved in mitotic exit. This suggested that LATS1 may be a component of the previously uncharacterized mitotic exit network in higher eukaryotes. Indeed, moderate overexpression of human LATS1 in cells exposed to microtubule poisons facilitated mitotic exit, and this activity required MOB1A. Reciprocally, small interfering RNA-mediated suppression of LATS1 or MOB1A prolonged telophase, but had no effect on the length of the earlier phases of mitosis. A role of LATS1 in mitotic exit may explain its previously described abilities to induce G2 arrest and promote cytokinesis.