Kinase-active Mutant Research Articles

Abstract 3888: DCLK1 is part of an EMT feedback loop and promotes colorectal cancer cell invasion and drug resistance

Abstract Colorectal cancer (CRC) is the third leading cause of cancer death in the U.S., with only a 6% 5-yr survival rate for stage IV disease. Its spread and acquisition of resistance to chemotherapy, which are fueled by the epithelial-mesenchymal transition (EMT) process and supported by tumor stem cells (TSCs), are major challenges to improving patient outcomes. New therapies that target stemness and EMT are desperately needed to prevent or delay metastasis and improve patient survival. Recently doublecortin-like kinase 1 (DCLK1) has been definitively proven to mark TSCs in CRC by two independent groups. Previous studies have demonstrated that DCLK1 is a prognostic factor in CRC and that targeted downregulation or inhibition of DCLK1 results in decreased CRC proliferation, migration, invasion, and other anti-oncogenic effects. However, the effect of overexpression of DCLK1 and its kinase active mutant on CRC has not been assessed. In this study, we investigate the correlative role of EMT and DCLK1 expression in CRC progress. Human colon cancer cells (HCT116) were infected with Lentivirus containing wild type DCLK1 or mutant DCLK1R326C cDNA sequences to overexpress DCLK1, DCLK1R326C or green fluorescent protein (GFP) cDNA sequence as control. The expressing levels of DCLK1 and EMT factors were analyzed by western blotting. The proliferative and invasive potential of these cells were compared using a MTT assay for proliferation, wound healing assay for migration, and Matrigel coated transwell assay for invasion. Knockdown of either ZEB1 or DCLK1 by specific siRNA in HCT116 cells was performed. The effects of siDCLK1 on 5-FU were performed in both HCT116 and SW480 cells using a Caspase 3/7 activity assay. Analysis of human CRC was performed using TCGA COADREAD dataset. Here we report that compared to GFP control cells, HCT116-DCLK1 and HCT116-DCLK1R326C cells exhibited a more than 20% increase in proliferation, approximately 30% increase in migration, and a 2-fold increase (p < 0.05) in invasion. DCLK1 expression level is decreased more than 30% by knocking down ZEB1 in HCT116 cells. In addition, knockdown DCLK1 increased 5-FU induced cell apoptosis more than 50% (P<0.05). Evidence from TCGA COADREAD demonstrated that EMT predicts survival in CRC patients, and increased expression level of DCLK1 in CRC patients correlate to EMT and mesenchymal phenotype. These data suggest that DCLK1 is a part of an EMT feedback-loop and may be exploited with DCLK1-targeted therapeutics for CRC. Citation Format: Dongfeng Qu, Nathaniel Weygant, William L. Berry, Randal May, Parthasarathy Chandrakesan, James J. Tomasek, Sripathi M. Sureban, Courtney Houchen, Yang Ge, Jiannan Yao, Guangyu An, Edwin Bannerman-Menson. DCLK1 is part of an EMT feedback loop and promotes colorectal cancer cell invasion and drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3888. doi:10.1158/1538-7445.AM2017-3888

Impaired Inflammatory Responses in Murine Lrrk2-Knockdown Brain Microglia

LRRK2, a Parkinson's disease associated gene, is highly expressed in microglia in addition to neurons; however, its function in microglia has not been evaluated. Using Lrrk2 knockdown (Lrrk2-KD) murine microglia prepared by lentiviral-mediated transfer of Lrrk2-specific small inhibitory hairpin RNA (shRNA), we found that Lrrk2 deficiency attenuated lipopolysaccharide (LPS)-induced mRNA and/or protein expression of inducible nitric oxide synthase, TNF-α, IL-1β and IL-6. LPS-induced phosphorylation of p38 mitogen-activated protein kinase and stimulation of NF-κB-responsive luciferase reporter activity was also decreased in Lrrk2-KD cells. Interestingly, the decrease in NF-κB transcriptional activity measured by luciferase assays appeared to reflect increased binding of the inhibitory NF-κB homodimer, p50/p50, to DNA. In LPS-responsive HEK293T cells, overexpression of the human LRRK2 pathologic, kinase-active mutant G2019S increased basal and LPS-induced levels of phosphorylated p38 and JNK, whereas wild-type and other pathologic (R1441C and G2385R) or artificial kinase-dead (D1994A) LRRK2 mutants either enhanced or did not change basal and LPS-induced p38 and JNK phosphorylation levels. However, wild-type LRRK2 and all LRRK2 mutant variants equally enhanced NF-κB transcriptional activity. Taken together, these results suggest that LRRK2 is a positive regulator of inflammation in murine microglia, and LRRK2 mutations may alter the microenvironment of the brain to favor neuroinflammation.

The c-Fes tyrosine kinase cooperates with the breakpoint cluster region protein (Bcr) to induce neurite extension in a Rac- and Cdc42-dependent manner

The c- fes locus encodes a cytoplasmic protein-tyrosine kinase (Fes) previously shown to accelerate nerve growth factor (NGF)-induced neurite outgrowth in rat PC12 cells. Here, we investigated the role of the Rho family small GTPases Rac1 and Cdc42 in Fes-mediated neuritogenesis, which have been implicated in neuronal differentiation in other systems. Fes-induced acceleration of neurite outgrowth in response to NGF treatment was completely blocked by the expression of dominant-negative Rac1 or Cdc42. Expression of a kinase-active mutant of Fes induced constitutive relocalization of endogenous Rac1 to the cell periphery in the absence of NGF, and led to dramatic actin reorganization and spontaneous neurite extension. We also investigated the breakpoint cluster region protein (Bcr), which possesses the Dbl and PH domains characteristic of guanine nucleotide exchange factors for Rho family GTPases, as a possible link between Fes, Rac/Cdc42 activation, and neuritogenesis. Coexpression of a GFP–Bcr fusion protein containing the Fes binding and tyrosine phosphorylation sites (amino acids 162–413) completely suppressed neurite outgrowth triggered by Fes. Conversely, coexpression of full-length Bcr with wild-type Fes in PC12 cells induced NGF-independent neurite formation. Taken together, these data suggest that Fes and Bcr cooperate to activate Rho family GTPases as part of a novel pathway regulating neurite extension in PC12 cells, and provide more evidence for an emerging role for Fes in neuronal differentiation.

Defective stress kinase and Bak activation in response to ionizing radiation but not cisplatin in a non-small cell lung carcinoma cell line

We have here examined ionizing radiation (IR)-induced apoptotic signaling in one IR-sensitive small cell lung carcinoma (SCLC) and one resistant non-small cell lung carcinoma (NSCLC) cell line, both harboring mutant p53. In the sensitive SCLC cell line, IR induced conformational modulation of Bak and Bax, mitochondrial depolarization, and nuclear fragmentation. These events were not observed in the IR-resistant NSCLC cell line. However, in the same cells, cisplatin, a DNA-damaging drug, induced Bak and Bax modulation, mitochondrial depolarization, and nuclear fragmentation. Pre-mitochondrial signaling events were examined in order to further characterize the differing IR response. In the SCLC cell line, IR-induced apoptotic signaling was found to involve a MEKK1-related pathway and activation of the stress-activated kinases JNK and p38. In comparison, the NSCLC cell line had higher basal levels of activity of JNK and p38, and IR treatment did not further activate these kinases. However, NSCLC cells were sensitive to Bak modulation and apoptosis induced by a kinase-active mutant of MEKK1. Together, the results delineate a mechanism of IR resistance in NSCLC cells and indicate that IR and cisplatin induce Bak modulation and apoptosis via different pathways.

Role of citron kinase as a target of the small GTPase Rho in cytokinesis.

During mitosis, a ring containing actin and myosin appears beneath the equatorial surface of animal cells. This ring then contracts, forms a cleavage furrow and divides the cell, a step known as cytokinesis. The two daughter cells often remain connected by an intercellular bridge which contains a refringent structure known as the midbody. How the appearance of this ring is regulated is unclear, although the small GTPase Rho, which controls the formation of actin structures, is known to be essential. Protein kinases are also thought to participate in cytokinesis. We now show that a splice variant of a Rho target protein, named citron, contains a protein kinase domain that is related to the Rho-associated kinases ROCK14 and ROK, which regulate myosin-based contractility. Citron kinase localizes to the cleavage furrow and midbody of HeLa cells; Rho is also localized in the midbody. We find that overexpression of citron mutants results in the production of multinucleate cells and that a kinase-active mutant causes abnormal contraction during cytokinesis. We propose that citron kinase regulates cytokinesis at a step after Rho in the contractile process.

Deletions in the SH2 Domain of p60v-srcPrevent Association with the Detergent-Insoluble Cellular Matrix

p60v-src has been shown to associate with a detergent-insoluble cellular matrix containing cytoskeletal proteins, but p60c-src does not bind to this matrix. We analyzed the association of mutant src proteins with the matrix and found that mutants which lack an amino-terminal portion (residues 149 to 169) of the SH2 domain cannot bind to the matrix. Neither the SH3 region nor other portions of the SH2 region were required for association. We also tested protein kinase-defective mutants and chimeras of p60v-src and p60c-src. We found a strong correlation between the kinase activity of p60src and its association with the detergent-insoluble matrix. Double infection of kinase-defective and kinase-active mutants did not result in matrix binding of the kinase-defective src proteins. We also found that Tyr-416, the major site of autophosphorylation in p60v-src, was not required for matrix association.

Deletions in the SH2 domain of p60v-src prevent association with the detergent-insoluble cellular matrix.

p60v-src has been shown to associate with a detergent-insoluble cellular matrix containing cytoskeletal proteins, but p60c-src does not bind to this matrix. We analyzed the association of mutant src proteins with the matrix and found that mutants which lack an amino-terminal portion (residues 149 to 169) of the SH2 domain cannot bind to the matrix. Neither the SH3 region nor other portions of the SH2 region were required for association. We also tested protein kinase-defective mutants and chimeras of p60v-src and p60c-src. We found a strong correlation between the kinase activity of p60src and its association with the detergent-insoluble matrix. Double infection of kinase-defective and kinase-active mutants did not result in matrix binding of the kinase-defective src proteins. We also found that Tyr-416, the major site of autophosphorylation in p60v-src, was not required for matrix association.