Fig 2: HA-CDKL3 co-immunoprecipitates endogenous Akt.Source data are available for this figure.

Fig 3: Protein mapping on cyclin-dependent kinase–like 3 (CDKL3).(A) Schematic drawing for CDKL FL (full-length), K (Kinase domain), ?K (Truncation of kinase domain) constructs. (B) Co-immunoprecipitation of Myc-Akt and HA-CDKL3 revealed that the kinase domain of CDKL3 is responsible for Akt interaction.Source data are available for this figure.

Fig 4: Cyclin-dependent kinase–like 3 (CDKL3) inhibits autophagy in osteosarcoma (OS).(A) An outline of OS clinical dataset analysis (GEO accession: GSE21257). 33 out of 53 patient samples were selectively analyzed based on the CDKL3 expression level for the differential gene expression patterns. (B) Signaling pathway was mapped by the R package clusterProfiler. Upper and bottom figures represent down-regulated and up-regulated pathways (CDKL3 top 0.3 versus bottom 0.3), respectively. KEGG enrichment analysis using the clusterProfiler R package was performed on differential expressed genes with a strict cutoff of P < 0.01 and false discovery rate of less than 0.05. The size of the dot indicates the number of differentially expressed genes in the pathway. Color of the dot represents the value of Benjamini and Hochberg false discovery rate–adjusted P-value. (C) Molecular connection of PI3K-Akt and AMPK pathway in regulation of cell growth, autophagy, etc. This summarized mechanism was extracted from the GEO dataset GSE21257. mTORC1 plays key roles in eukaryotic cell metabolism by promoting cell growth and inhibiting autophagy and apoptosis. (D) Representative images of parental cells and CDKL3-KO U2OS and Saos-2 cells transfected with mRFP-eGFP-LC3, at normal conditions (DMEM with 10% FBS, control) or 6 h EBSS treatment (starvation). Colocalized mRFP and eGFP (yellow dots) indicate autophagosomes, only mRFP (red dots) indicates autolysosomes. Nuclei (blue) were labeled with DAPI. Scale bar = 5 µm. (E) Quantification of autophagosomes (eGFP+mRFP+; yellow) and autolysosomes (eGFP-mRFP+; red) per cell from (D) (n = 3). (F) Western blot analysis of LC3B, Beclin-1, and Atg5-Atg12 in indicated cells. The number below each blotting strip is intensity quantification derived from ImageJ. Error bars indicate SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test.Source data are available for this figure.

Fig 5: TAL1 expressing pro-T cells show a growth disadvantage, which can be rescued by co-expressing mutant AKT. (A) Scheme of ex vivo pro-T-cell culture derived from Cre-ER mice, requiring interleukin-7 (IL7), stem cell factor (SCF) and immobilized Delta-like ligand 4 (DLL4) for proliferation. (B) Pro-T cells were transduced with inducible retroviral constructs for expression of TA L1, AKTE17K or TAL1+AKTE17K. Pro-T cells were sorted for green fluorescent protein (GFP) and treated with 1 mM 4-OH tamoxifen for 48 hours to activate Cre-ER and induce LoxP-mediated inversion and expression of the oncogenes. Constructs having TAL1 and AKTE17K become blue fluorescent protein (BFP)-positive, indicating the construct has successfully flipped. The construct containing both TAL1 and AKTE17K does not contain BFP. (C) The percentages of each population in pro-T cells having TAL1 or TAL1+AKTE17K were followed over time (BFP) cells when co-expression with mutant AKT (mcherry). Co-transduced cells in which the constructs were flipped become the largest population (BFP + mcherry). (D) Cell densities (mean +/- standard deviation) over time for different proT-cells conditions. (E) Cell densities (mean with standard deviation), as measure of absolute proliferation, were measured over time for different pro-T cell conditions: TA L1, AKTE17K, TAL1+AKTE17K and WT empty control. F, TA L1-AKTE17K expressing pro-T cells were able to grow in the absence of IL7.