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Considered one of the frequently deregulated pathways in leukemia, the PI3K/AKT pathway is a highly conserved signal transduction pathway associated with cell growth, survival, and proliferation. When phosphatidylinositol 3-kinase (PI3K) is activated, it phosphorylates lipids on the plasma membrane, leading to conversion of secondary messenger phosphatidylinositol (4,5)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Protein kinase B (PKB or AKT) binds PIP3, which allows a master kinase effector 3-phosphoinositide-dependent protein kinase-1 (PDK1) to access and phosphorylate AKT at its activation loop threonine 308, resulting in its partial activation. Currently, PDK1 is the only known kinase that is able to mediate this phosphorylation, which is essential for full AKT activity. Phosphatase and tensin homolog (PTEN) antagonizes the pathway by converting PIP3 back to PIP2. Loss of PTEN leads to excessive PIP3 build-up at the plasma membrane, which in turn, recruits and activates AKT, causing uncontrolled activity in the PI3K/AKT pathway downstream. Using inducible leukemic mouse models, we were able to investigate the intricate relationship between PTEN and PDK1 further. Upon transplantation, we observed that when PTEN was absent, there was no effect on engraftment when compared to the control. However, it did result in early onset of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), biphenotypic acute leukemia (BAL), and mixed-lineage leukemia (MLL). On the contrary, when PDK1 was deleted, it did not lead to leukemia development over time. However, when both PDK1 and PTEN were knocked out simultaneously, the disease latency increased, but it still was not able to reverse leukemogenesis altogether. Interestingly, these mice only succumbed to myeloid malignancy and not lymphoid leukemia. The presence of AKT phosphorylation at T308 was observed in the double knockout animals upon disease development but was absent before disease onset. From this, we hypothesized that there might be an alternative kinase or mechanism that is able to phosphorylate AKT, leading to disease progression of acute myeloid leukemia. Approaches such as RNA sequencing, high-throughput inhibitor screens, and proteomic profiling competitive assays were taken in order to explore the possibility of another kinase that could be involved in this functional crosstalk. As a result, we concluded that PDK1 is required for PTEN-mediated leukemia and that there might be other kinases or pathways that can contribute to the activation of AKT in the absence of PDK1.

e big part of biomass is in the form of plant cell walls. Fungi play an essential role in carbon and nitrogen recycling, in which one of the means is by degrading polysaccharides that human cannot utilize efficiently. To get a deeper insight into degradation of pectin, one of the major groups of plant polysaccharides six polygalacturonases, one rhamnogalacturonase, and one xylogalacturonase from a thermophilic fungus, Aspergillus niveus found widely in soil and decaying organic matter, were cloned and expressed in Aspergillus nidulans fused to a his tag. These enzymes break down different components of pectin. Their functions and activities are studied by cloning using E. coli , expression using A. nidulans , purification using Ni-NTA, and activity determination using various substrates. Western Blotting detected that the His-tag was expressed correctly. However, purification using Ni-NTA failed, indicating the His-tag was not able to bind to Ni resin. SDS-PAGE protein gels showed the actual sizes of the proteins were larger than theoretical sizes. Expression and purification studies suggested that all eight enzymes expressed in A. nidulans were probably highly glycosylated. Pectic acid was used as substrate for the six polygalacturonases; rhamnogalacturonan from citrus pectin was used for the rhamnogalacturonase and partially hydrolyzed gum tragacanth was used for the xylogalacturonase. Only one exo-polygalacturonase does not have signal peptide. Among eight enzymes, three of six polygalacturonases are exo-polygalacturonases and the others are endo-polygalacturonases. For the exo-polygalacturonases, all of them cut off one or two GalA at a time. One has some limitation on length of substrate. The specific limitation remains unknown. For the endo-polygalacturonases, all of them work in endo-style. Final products from pectic acid for one of them were GalA1-GalA4. For another they were only GalA1 and GalA2 while for the third the main products were GalA1-GalA5. For the rhamnogalacturonase, the final products were RG2 and RG3. And for the xylogalacturonase, the information about final products is unavailable at present. NMR will be performed for their identification.