1. LATS 1/2 (Large Tumor Suppressor 1 & 2)
By: Carly Speight
BIOL 445
In mammalian systems, the Large Tumor Suppressor (LATS) family of proteins has increasingly been shown to be an important regulator in cellular homeostasis. Because of their high degree of homology and significant overlap in function, LATS 1 and 2 comprise a unique signaling family within the cell.

2. LATS1 and LATS2 share several conserved domains including the C-terminal Ser/Thr kinase domain, whereas the N-terminal domain varies significantly and accounts for functional differences
The lats gene was first identified in Drosophila in 1995 by a research group at Yale University in efforts to search for tumor suppresser genes in genetic mosaics. Tissue specific loss led to overgrowth and tumor formation, thus confirming their identification of a tumor suppressor.
Since it’s initial discovery in Drosophila in 1995, LATS has been shown to be evolutionarily conserved with homologs also found in budding yeast, fission yeast, and C. elegans, mice, and humans.
The LATS-encoded proteins belong to the AGC family of serine/threonine kinases. Other features of the LATS gene include:
A protein binding domain (PBD) that lies upstream of the catalytic domain and binds several proteins including MOB (scaffold protein) and LIMK1
Two LATS conserved domains LCD1 and LCD2 that are essential and critical for the tumor suppresser’s activity (deletions of either in mouse LATS2 abolished its tumor suppressor activity)
A PPxY (P=proline, x=any amino acid, Y=tyrosine) motifs that bind proteins such as YAP and TAZ
A ubitquitin-associated domain (UBA) on the N-terminal domain.
As individual proteins, LATS1 possesses a region of proline residues (called P-stretch) downstream of the UBA that assists in protein-protein interaction, and LATS2 possesses a PA repeat (alternating proline/alanine residues)
Meng, et.al. 2016

3. The core Hippo kinase cascade begins with activation of MST1/2, which phosphorylates LATS1/2, and ultimately restricts activation of YAP/TAZ
The physiological output of this cascade is to restrict the activities of two transcriptional co-activators: YAP and TAZ. When YAP and TAZ are active, they translocate into the nucleus to bind the TEAD transcription factor family and induce expression of a wide variety of genes that are involved in cell proliferation, survival and migration.
When the pathway is inactive, YAP and TAZ are unphosphorylated and located in the nucleus
The Hippo pathway can be activated by TAO kinases, which phosphorylate MST1/2 at its activation loop
- it is possible that MST activation can be initiated by dimerization caused by autophosphorylation
MST1/2 in turn phosphorylates LATS1/2, facilitated by various scaffold proteins (a few involved are SAV1, MOB, and NF2) at its hydrophobic motifs
MAP4K1/2/3/5 and MAP4K4/6/7 (requires NF2) can also directly phosphorylate LATS1/2 at their hydrophobic motifs
Activated LATS1/2 phosphorylates YAP/TAZ and results in cytoplasmic retention and degradation
Visser & Yang (2010)

4. LATS activity can be regulated by precise subcellular localization, allowing for specific activation and direct targeting of proteins
Several mechanisms exist for the regulation of LATS: regulation of expression at both the transcriptional and protein level, post-translational modifications such as phosphorylation and ubitquitination, control of localization (focusing on this) and conformational regulation.
Figure on top: MOB1 recruits LATS1 to the plasma membrane where it can be activated by MST1/2. Conversely, mutant LATS1 that are unable to bind MOB1 cannot translocate to the membrane and cannot be phosphorylated.
Figure on bottom:
Once activated, LATS can travel to other parts of the cell and be recruited by other proteins to perform specific functions. One for example is Aurora-A, a centrosomal kinase, which is responsible for phosphorylating LATS2 on Ser83 during mitosis and localizing LATS2 in the centrosome during the cell cycle
The Ser83 residue is conserved in human LATS1/2 and also in mouse LATS1/2
To confirm that Ser83 of LATS2 is phosphorylated by Aurora-A, a experiment was conducted using mouse models that raised a specific monoclonal mouse antibody (3B11) by immunizing mice with a LATS2 peptide whose Ser83 residue was phosphorylated
Results: when WT Aurora-A was used, the 3B11 antibody specifically detected the phosphorylated form of LATS (lane 2)
No phosphorylation of the Ser83 site was detected with kinase-dead Aurora-A was used (lane 3)
Therefore: the 3B11 antibody is specific only for the phosphorylated form of Ser83
Meng, et. Al (2016)
Toji, et. Al (2004)

5. LATS2 co-localizes with Aurora-A at the centrosome during mitosis
Hypothesis: if LATS2 is a phosphorylation target of Aurora-A in vivo, then it is likely that their distributions are similar during the cell cycle
HeLa S3 cells were transfected with GFP-fused LATS2
The transfected cells were synchronized at the S phase by thymidine-single block, released from the block, and then fixed with formaldehyde at various cell cycle stages:
Interphase  i, ii
Prophase  iii
Metaphase  iv
Anaphase is not shown in this figure
Telophase  v
LATS2 is visualized with green immunoflorescent staining and Aurora-A and centrosomes are visualized with red
Yellow signals indicate the co-localization of GFP-tagged LATS2 and Aurora-A at the centrosomes (during interphase, prophase, and telophase)
Toji, et. Al. (2004)

6. By inhibiting kinase activity of two essential cyclin kinase complexes, LATS1 and LATS2 arrest cells in either G1/S phase or G2/M phase and inhibit cell proliferation
As expected, any changes in proliferation are correlated with changes in the cell cycle
It was demonstrated in the figure on the right that LATS1 overexpression causes G2/M arrest or apoptosis in human cancer cells
Method for part A:
Infected HeLa cells with LATS1 adenovirus at increasing multiplicity of infection (MOI)
Western blots show that level of LATS1 expression is progressively enhanced with increasing virus infection
Part B: Increasing LATS1 leads to an increase in the percentage of HeLa cells in G2/M and a corresponding decrease of HeLa cells in G1/S
Since the G2/M transition is primarily controlled by the Cyclin A/B / Cdc2 complex, they showed that overexpression of LATS1 inhibits the kinase activity of the complex
** It is important to keep in mind that these protein complexes (E/CDK2, A/B/CDC2) cannot be phosphorylated by LATS, only negatively regulated; YAP and TAZ are the only known substrates of LATS identified so far
Yang, e. al. (2001)

7. In a study of breast cancer patients, low LATS1 expression due to promoter hyper-methylation has been associated with a biologically aggressive phenotype
Loss of function of LATS1 and 2 is unlikely to be induced by the combination of somatic mutation and LOH, but is more likely to be induced by other mechanisms, such as hyper-methylation (increased prevalence of CpG islands).
In a study of breast cancer tissues, the LATS1 promoter was found to be hyper-methylated in 17/30 tumors (56.7%) causing a significant decrease in LATS1 mRNA expression
In patients, decreased LATS mRNA expression was associated with large tumor size, lymph node metastasis, and estrogen and progesterone negativity.
Thus, loss of LATS1 expression in these cases is associated with a poor prognosis.
Figure on right showing levels of LATS1/2 mRNA expression : M = hyper-methylated, UM = without hyper-methylation
Takahashi, et. Al. (2005)

8. Treatment of cells with nocodazole, which disrupts the centrosome and mitotic checkpoints, causes nuclear localization and activation of p53-dependent events
Nocodazole
LATS2 appears to activate the G1 checkpoint through p53 (there is a p53 response element in the promoter region of LATS2)
At the transcriptional level, p53 has been shown to induce LATS2 mRNA levels in response to nocodazole
In response to nocodazole, LATS2 was shown to stabilize p53 by binding and inhibiting Mdm2
- Nocodazole-treated cells cannot form metaphase spindles because the microtubules cannot polymerize (no addition of tubulin)
Nocodazole induces LATS2 translocation from centrosomes to the nucleus and p53 accumulation
In turn, p53 selectively upregulates LATS2 expression in G2/M phase cells – defining the positive feedback loop
* the positive feedback loop between p53 and the LATS2 tumor suppressors prevents tetraploidization
Ensures proper function of the G1 tetraploidy checkpoint
Taxol – binds tightly to polymers and inhibits microtubule depolymerization – ensures proper function of the G1 tetraploidy checkpoint
There is a large fraction of human cancers mutated in the p53 gene, suggesting a wide therapeutic applicability
There is also therapeutic applicability in promoting the coupling of pre-mitotic cell cycle progression to microtubule integrity in some breast cancer cell lines
Taxol

9. References http://www.tandfonline.com/doi/abs/10.4161/cc.9.19.13386
Stacy Visser & Xiaolong Yang (2010) LATS tumor suppressor: A new governor of cellular homeostasis , Cell Cycle, 9:19, , DOI: /cc
Zhipeng Meng, Toshiro Moroishi, and Kun-Liang Guan. (2016) Mechanisms of Hippo Pathway Regulation. Cold Spring Harbor Laboratory Press.