Breast tumor kinase BRK requires kinesin-2 subunit KAP3A in modulation of cell migration

BReast tumor Kinase (BRK) also known as protein kinase 6 (PTK6) is a nonreceptor tyrosine kinase overexpressed in the majority of human breast tumors. Although some studies have implicated BRK in signalling, cell proliferation and migration, the precise intracellular role of BRK has not been fully elucidated. The RNA-binding protein Sam68, and adaptor proteins paxillin and STAT3 are the only BRK substrates that link BRK to signal transduction. To identify new BRK substrates, we screened high-density protein filter arrays by large-scale in vitro kinase assays using active recombinant BRK. We identified at least 4 BRK targets comprising the alpha-subunit of stimulatory guanine nucleotide binding protein (GNAS), FL139441, beta-tubulin and kinesin associated protein 3A (KAP3A) and validated them as BRK substrates using a secondary assay. Further characterization revealed that KAP3A is an in vivo substrate of BRK and associates with BRK in breast cancer cells. We show that BRK specifically phosphorylated tyrosine residues at the C-terminus of KAP3A and induces delocalization of KAP3A from punctate nuclear localization to a diffuse nucleo-cytoplasmic pattern. Functionally, we demonstrate that KAP3A knockdown results in suppression of BRK-induced migration of breast cancer cells and show that the C-terminal deletion mutant of KAP3A acts as a dominant negative in BRK-induced cell migration. Our findings therefore reveal new substrates of BRK and define KAP3A as a physiological substrate of BRK during cell migration.     

Genomic organization and DNA sequence of the mouse kidney androgen-regulated protein (KAP) gene

The gene for kidney androgen-regulated protein (KAP) is expressed under androgenic control in the epithelial cells of the renal cortical proximal tubules. However, there is an androgen-independent component of the expression of this gene that occurs specifically in the outermedullary S3 segments of the proximal tubules. In these cells, the KAP gene is estrogen responsive and its expression is dependent on pituitary function. As a first step in correlating its interesting cell-specific and hormonal regulation with the structure of the gene, the genomic organization of the KAP gene was described and sequence of the gene and the proximal 1 kb of 5'-flanking DNA was determined. Sequence motifs were identified in the 5'-flanking DNA that may function in the regulation KAP gene expression by androgen, estrogen, and pituitary glycoprotein hormones. The gene is present in a single copy in the mouse genome and is 3,807 nucleotides in length. It contains 4 exons of 120, 177, 63, and 251 nucleotides and three intervening sequences of 1,450, 126, and 1,620 nucleotides. The gene exhibits a high degree of a genetic polymorphism as revealed by comparison of restriction digests of DNA from two highly inbred strains, BALB/c and C57BL/6.     

KAP1 dictates p53 response induced by chemotherapeutic agents via Mdm2 interaction

KAP1 recruits many proteins involved in gene silencing and functions as an integral part of co-repressor complex. KAP1 was identified as Mdm2-binding protein and shown to form a complex with Mdm2 and p53 in vivo. We examined the role of KAP1 in p53 activation after the treatment of cells with different types of external stresses. KAP1 reduction markedly enhanced the induction of p21, a product of the p53 target gene, after treatment with actinomycin D or gamma-irradiation, but not with camptothecin. Treatment with actinomycin D, but not with camptothecin, augmented the interaction of p53 with Mdm2 and KAP1. Further, KAP1 reduction in actinomycin D-treated cells facilitated cell cycle arrest and negatively affected clonal cell growth. Thus, the reduction of KAP1 levels promotes p53-dependent p21 induction and inhibits cell proliferation in actinomycin D-treated cells. KAP1 may serve as a therapeutic target against cancer in combination with actinomycin D.     

Androgen Suppresses the Proliferation of Androgen Receptor-Positive Castration-Resistant Prostate Cancer Cells via Inhibition of Cdk2, CyclinA,and Skp2

The majority of prostate cancer (PCa) patient receiving androgen ablation therapy eventually develop castration-resistant prostate cancer (CRPC). We previously reported that androgen treatment suppresses Skp2 and c-Myc through androgen receptor (AR) and induced G1 cell cycle arrest in androgen-independent LNCaP 104-R2 cells, a late stage CRPC cell line model. However, the mechanism of androgenic regulation of Skp2 in CRPC cells was not fully understood. In this study, we investigated the androgenic regulation of Skp2 in two AR-positive CRPC cell line models, the LNCaP 104-R1 and PC-3^AR Cells. The former one is an early stage androgen-independent LNCaP cells, while the later one is PC-3 cells re-expressing either wild type AR or mutant LNCaP AR. Proliferation of LNCaP 104-R1 and PC-3^AR cells is not dependent on but is suppressed by androgen. We observed in this study that androgen treatment reduced protein expression of Cdk2, Cdk7, Cyclin A, cyclin H, Skp2, c-Myc, and E2F-1; lessened phosphorylation of Thr14, Tyr15, and Thr160 on Cdk2; decreased activity of Cdk2; induced protein level of p27^Kip1; and caused G1 cell cycle arrest in LNCaP 104-R1 cells and PC-3^AR cells. Overexpression of Skp2 protein in LNCaP 104-R1 or PC-3^AR cells partially blocked accumulation of p27^Kip1 and increased Cdk2 activity under androgen treatment, which partially blocked the androgenic suppressive effects on proliferation and cell cycle. Analyzing on-line gene array data of 214 normal and PCa samples indicated that gene expression of Skp2, Cdk2, and cyclin A positively correlates to each other, while Cdk7 negatively correlates to these genes. These observations suggested that androgen suppresses the proliferation of CRPC cells partially through inhibition of Cyclin A, Cdk2, and Skp2.     

Binding of HTm4 to cyclin-dependent kinase (Cdk)-associated phosphatase (KAP).Cdk2.cyclin A complex enhances the phosphatase activity of KAP, dissociates cyclin A, and facilitates KAP dephosphorylation of Cdk2

Cyclin-dependent kinase 2 (cdk2) activation requires phosphorylation of Thr160 and dissociation from cyclin A. The T-loop of cdk2 contains a regulatory phosphorylation site at Thr160. An interaction between cdc-associated phosphatase (KAP) and cdk2 compromises the interaction between cdk2 and cyclin A, which permits access of KAP, a Thr160-directed phosphatase, to its substrate, cdk2. We have reported that KAP is bound and activated by a nuclear membrane protein, HTm4. Here, we present in vitro data showing the direct interaction between the HTm4 C terminus and KAP Tyr141. We show that this interaction not only facilitates access of KAP to Thr160 and accelerates KAP kinetics, but also forces exclusion of cyclin A from the KAP.cdk2 complex.     

Inhibition of SIRT6 in prostate cancer reduces cell viability and increases sensitivity to chemotherapeutics

SI RT6 is an important histone modifying protein that regulates DNA repair, telomere maintenance, energy metabolism, and target gene expression. Recently SIRT6 has been identifi ed as a tumor suppressor and is downregulated in certain cancer types, but not in other cancers. From deposited gene profi ling studies we found that SIRT6 was overexpressed in prostate tumors, compared with normal or paratumor prostate tissues. Tissue microarray studies confi rmed the higher levels of SIRT6 in both prostate tumor tissues and prostate cancer cells than in their normal counterparts. Knockdown of SIRT6 in human prostate cancer cells led to sub-G1 phase arrest of cell cycle, increased apoptosis, elevated DNA damage level and decrease in BCL2 gene expression. Moreover, SIRT6-deficiency reduced cell viability and enhanced chemotherapeutics sensitivity. Taken together, this study provides the fi rst evidence of SIRT6 overexpression in human prostate cancer, and SIRT6 regulation could be exploited for prostate cancer therapy.     

Characterization and expression analysis of KAP7.1, KAP8.2 gene in Liaoning new-breeding cashmere goat hair follicle

Keratin-associated protein is one of the major structural proteins of the hair, whose content in hair has important effect on the quality of cashmere. In order to study the relationship between HGTKAP gene expression and cashmere fineness, the quantitative real-time RT–PCR (qRT–PCR) was firstly used to detect the levels of KAP7.1, KAP8.2 gene expression in the primary and secondary hair follicles; semi-quantitative RT–PCR was used to detect whether KAP7.1, KAP8.2 gene are expressed in heart, liver, spleen, lung, kidney tissues; and in situ hybridization(ISH) to detect KAP7.1 gene expression location. qRT–PCR result showed that the expression of both KAP7.1 and KAP8.2 gene in the secondary hair follicles are significantly higher than that in the primary follicles, relative quantitative analysis obtained that KAP7.1 was 2.28 times, while KAP8.2 was 2.71 times. Semi-quantitative RT–PCR results revealed that KAP 7.1 and KAP8.2 mRNA were not detected in the heart, liver, spleen, lung and kidney tissues, demonstrating that KAP7.1 and KAP8.2 were specially expressed in hair follicles, participating in hair formation. Moreover, KAP7.1 gene has a strong expression in the cortical layer, inner root sheath of the primary follicles and the cortical layer, inner root sheath and hair matrix of the secondary hair follicles by ISH analysis. Taken together, the evidence presented here indicated that in the formation of cashmere and wool, differential expression of these two genes in the primary and secondary hair follicles may have an important role in regulating the fiber diameter.     

Expression of the protein phosphatase 1 inhibitor KEPI is downregulated in breast cancer cell lines and tissues and involved in the regulation of the tumor suppressor EGR1 via the MEK-ERK pathway

KEPI is a protein kinase C-potentiated inhibitory protein for type 1 Ser/Thr protein phosphatases. We found no or reduced expression of KEPI in breast cancer cell lines, breast tumors and metastases in comparison to normal breast cell lines and tissues, respectively. KEPI protein expression and ubiquitous localization was detected with a newly generated antibody. Ectopic KEPI expression in MCF7 breast cancer cells induced differential expression of 95 genes, including the up-regulation of the tumor suppressors EGR1 (early growth response 1) and PTEN (phosphatase and tensin homolog), which is regulated by EGR1. We further show that the up-regulation of EGR1 in MCF7/KEPI cells is mediated by MEK-ERK signaling. The inhibition of this pathway by the MEK inhibitor UO126 led to a strong decrease in EGR1 expression in MCF7/KEPI cells. These results reveal a novel role for KEPI in the regulation of the tumor suppressor gene EGR1 via activation of the MEK-ERK MAPK pathway.     

Vitamin D inhibits G1 to S progression in LNCaP prostate cancer cells through p27Kip1 stabilization and Cdk2 mislocalization to the cytoplasm

1,25-(OH)2 vitamin D3 (1,25-(OH)2D3) exerts antiproliferative effects via cell cycle regulation in a variety of tumor cells, including prostate. We have previously shown that in the human prostate cancer cell line LN-CaP, 1,25-(OH)2D3 mediates an increase in cyclin-dependent kinase inhibitor p27Kip1 levels, inhibition of cyclin-dependent kinase 2 (Cdk2) activity, hypophosphorylation of retinoblastoma protein, and accumulation of cells in G1. In this study, we investigated the mechanism whereby 1,25-(OH)2D3 increases p27 levels. 1,25-(OH)2D3 had no effect on p27 mRNA levels or on the regulation of a 3.5-kb fragment of the p27 promoter. The rate of p27 protein synthesis was not affected by 1,25-(OH)2D3 as measured by luciferase activity driven by the 5'- and 3'-untranslated regions of p27 that regulate p27 protein synthesis. Pulse-chase analysis of 35S-labeled p27 revealed an increased p27 protein half-life with 1,25-(OH)2D3 treatment. Because Cdk2-mediated phosphorylation of p27 at Thr187 targets p27 for Skp2-mediated degradation, we examined the phosphorylation status of p27 in 1,25-(OH)2D3-treated cells. 1,25-(OH)2D3 decreased levels of Thr187 phosphorylated p27, consistent with inhibition of Thr187 phosphorylation-dependent p27 degradation. In addition, 1,25-(OH)2D3 reduced Skp2 protein levels in LNCaP cells. Cdk2 is activated in the nucleus by Cdk-activating kinase through Thr160 phosphorylation and by cdc25A phosphatase via Thr14 and Tyr15 dephosphorylation. Interestingly, 1,25-(OH)2D3 decreased nuclear Cdk2 levels as assessed by subcellular fractionation and confocal microscopy. Inhibition of Cdk2 by 1,25-(OH)2D3 may thus involve two mechanisms: 1) reduced nuclear Cdk2 available for cyclin binding and activation and 2) impairment of cyclin E-Cdk2-dependent p27 degradation through cytoplasmic mislocalization of Cdk2. These data suggest that Cdk2 mislocalization is central to the antiproliferative effects of 1,25-(OH)2D3.     

Identification of protein-tyrosine phosphatase 1B as the major tyrosine phosphatase activity capable of dephosphorylating and activating c-Src in several human breast cancer cell lines

c-Src tyrosine kinase activity is elevated in several types of human cancer, and this has been attributed to elevated c-Src expression levels, increased c-Src specific activity, and activating mutations in c-Src. We have found a number of human breast cancer cell lines with elevated c-Src specific activity that also possess elevated phosphatase activity directed against the carboxyl-terminal negative regulatory domain of Src family kinases. To identify this phosphatase, cell extracts from MDA-MB-435S cells were chromatographed and the fractions were assayed for phosphatase activity. Four peaks of phosphatase activity directed against the nonspecific substrate poly(Glu/Tyr) were detected. One peak also dephosphorylated a peptide modeled against the c-Src carboxyl-terminal negative regulatory domain and intact human c-Src. Immunoblotting and immunodepletion experiments identified the phosphatase as protein-tyrosine phosphatase 1B (PTP1B). Examination of several human breast cancer cell lines with increased c-Src activity showed elevated levels of PTP1B protein relative to normal control breast cells. In vitro c-Src reactivation experiments confirmed the ability of PTP1B to dephosphorylate and activate c-Src. In vivo overexpression of PTP1B in 293 cells caused a 2-fold increase of endogenous c-Src kinase activity. Our findings indicate that PTP1B is the primary protein-tyrosine phosphatase capable of dephosphorylating c-Src in several human breast cancer cell lines and suggests a regulatory role for PTP1B in the control of c-Src kinase activity.