Tankyrases (TANK1 and TANK2) IRAP (Insulin responsive Amino peptidase, GBR14, and Tab182 
 

The 3'ends of chromosomes are capped with telomere sequences (TTAGGG; 6-26 nucleotides in length) by ribonucleoprotein telomerase during DNA replication. Telomerase is an unusual RNA-dependent DNA polymerase that uses and RNA component to specify the addition of telomere. The telomeric RNA contains a sequence complementary to TTAGGG. Approx. 4.8 kb of telomeric DNA is lost with each cell division. Many mammalian cells do not express telomerase resulting into shortening of telomere with each cell division, and ultimately causing the chromosomal instability, aging and cell death. Interesting, most transformed, immortalized or tumor cells continue to express telomerase. Introduction of telomerase into normal human cells has been shown to extend normal cell life by ~ 20 doubling.

Poly(ADP-ribose) polymerases (PARPs) catalyze formation of long, branched chain of poly(ADP-ribose) onto protein acceptors using NAD+ as a substrate. Poly(ADP)ribosylation is a transient posttranslational modification that can either enhance or reduce protein activity. Tankyrase (TRF1 interacting ankyrin-related ADP-ribose polymerase; human 1327 aa, renamed as TANK1, chromosome 8), a modular protein with homology to ankyrin and poly(adenosine diphosphate-ribose) polymerase (PARP) has been cloned and localized to telomere. The N-terminal HPS domain contains multiple run of histidine, proline, and serine residue homopolymers. TANK1 has 24 ankyrin repeats in TRF-1 interacting domain near the N-terminus. The 33-aa ANK repeat motif mediates protein-protein interactions. The ANK domain is followed another protein interaction motif called the sterile alpha-module (SAM). The C-terminal region of TANK1 contains the PARP activity. TANK1 uses its ANK domain to bind TRF1 and its PARP domain to ADP-ribosylate itself and TRF1, and thereby inhibiting the ability of TRF1 to bind telomere. The homology between tankyrase and PARPs is limited to catalytic domain. Takyrase-1 is expressed in many tissues and targeted to various intracellular compartments. Tanyrase-1, devoid of NLS (nuclear localization signal), is translocated to telomere (nucleus) through binding of its ANK domain to TRF1.

More recently a second tankyrase termed tankyrase-2/TANK2/TNKL, has been cloned and characterized. TANK2 gene (chromosome 10q23.2) encodes a ~130-kDa, 1166 aa protein with ~83% identity with TANK1. TANK2 has a unique N-terminus and lacks the HPS domain found in TANk1. TANK2 interacted with TRF1 and displayed APRP activity. It is widely expressed with more abundant expression in skeletal muscle and placenta. Unlike TANK1, TANk2 over expression caused rapid cell death.

In addition to regulating the telomere and nuclear localization, TANKs also resides at the other subcellular compartments such as nuclear envelope, specifically on cytoplasmic fibers of nuclear pore complexes. TANk1 is targeted to pericentriolar domain of centrosomes during mitosis. After mitosis, TANK1 associates with the golgi as a peripheral membrane protein and implicated in targeting of GLUT4 vesicles. In the absence of insulin, GLUT4 vesicles reside in the Golgi and cytoplasm. Upon stimulation by insulin, GLUT4 vesicles undergo exocytosis and translocate to the plasma membrane. GLUT4 vesicles are characterized by the presence of glucose transporter 4 (GLUT4) and IRAP (Insulin regulated aminopeptidase). The exocytosis of GLUT4 vesicles is therefore plays an important role in uptake of glucose through Glut4 and the degradation of various vascoactive hormones by IRAP. TANKs have been colocalized in GLUT4 vesicles and shown to bind IRAP via the ANK-repeats. IRAP (also known as placental leucine aminopeptidase, PLAP; leucyl cystinyl aminopeptidase, LNPEP; oxytocinase, Otase; vesicle protein of 165 kDa or vp165) belongs to the peptidase M1 family. IRAP (mouse 966aa, rat 1025 aa; human 1025 aa, chromosome 5q14.2-q15) release an n-terminal amino acid, cleave before cysteine, leucine as well as other amino acids. IRAP, a zinc-containing metalloprotein, degrade peptide hormone such as oxytocin, vasopressin, and angiotensin III, inactivate Met-enkephalin and dynorphin. It is a type 2 membrane protein and also secreted due to proteolytic processing. IRAP is glycosylated and alternatively spliced to at least 3 isoform (1-15 aa missing in isoform 2 and 1-20 missing in isoform 3). The N-terminus (1-110 aa) is located in the cytoplasm, followed by TM domain (111-131 aa) and the C-terminus (132-1025 aa) is extracellualr. The secreted form is cleaved at 154-155 aa (processed secreted form 155-1025 aa). IRAP is highly expressed in placenta, heart, kidney, and small intestine. In brain, only the membrane form is found. The brain form is found to be 140 kDa due to differential glycosylation.

TANK2 has also been shown to reside in the low-density microsomes and associate with the adapter protein, Grb14. The N-terminal 110-aa of Grb14 and 10-19 ANK-repeats of TANK2 mediate this interaction. GRB14 (mouse/rat 538 aa, human 540 aa, chromosome 2) is a member of GRB7 (Growth receptor bound proteins) family that includes Grb7, and Grb10. Grb family is characterized by the presence of a conserved, short non-catalytic SH2 domain (Src homology region 2) that binds to peptide sequences containing phopshotyrosine. Many intracellular targets of receptor tyrosine kinases contain 1 or more SH2 domains. GRB14 is widely distributed in many tissues with an abundance in testis, ovary, heart, liver, skeletal muscle, kidney, and pancreas. TANKs may play a role vesicle trafficking and in cytoplasmic signal transduction pathways via the Grg14.

Recently a novel TANK1-binding protein termed TAB182 (tankyrase-binding protein of 182 kDa), which binds to the ANK domain of TANK1 and TANK2 has been identified. TAB182 (human 1729 aa, predicted size 182 kDa, actual size ~200 kDa) is expressed in heart, lung, liver, kidney, pancreas, testing, ovary etc. TAB182 localizes to nucleus and to the cytoplasm, where it co localizes with cortical actin network. It serves as an acceptor of poly(ADP-ribosyl)ation by TANK1. TANK1 is suggested to act as scaffold for large molecular mass complexes made up of multiple binding proteins.

 

List of publications using ADI's Telomerase/Est2:

Telomerase/Est2 Zhang P 2003 FASEB J Feb 2003 in press TERT suppresses apoptotis at a premitochondrial step by a mechanism requiring reverse transcriptase activity and 14-3-3 protein binding ability,
IHC used 1:3K; also AIF/mab.

Telomerase/Est2 Hiyama E 2003 Cancer Lett. in press Telomerase as tumor marker IHC bronchial biopsy sample obtained from a patient with squamous cell carcinoma of the lung/EST21-A.

TRF2 Oh H 2003 Telomere attrition and Chk2 activation in human heart failure PNAS, Apr 2003; 100: 5378 - 5383. WB IHC rat.

Telomerase/Est2 Hiyama, E et al 2001 Neoplasia, 2001, 3, 17-26 Immunohistochemical detection of telomerase (hTERT) protein in human cancer tissues and a subset of cells in normal tissues paraffin section, est21-c.

Telomerase/Est2 Xiang Hua et al 2000 BBRC 278, 503-510, hTERT Can Function with Rabbit Telomerase RNA: Regulation of Gene Expression and Attenuation of Apoptosis.

Notes: Antibodies usage is indicated in the following techniques: WB=Western Blot ; IHC-Immunohistochmistry; IP=Immunoprecipition; Flow=Flow cytometry; Rev. 30626


m=mouse; r=rat; h=human; b=bovine; d=dog;ch=chicken; ~CT or ~NT=near C or N-terminus. EC=Extracellular; CP=Cytoplasmic domain; I=Internal;

* Expected antibody crossreactivity information is mostly based upon high (>70%) sequence conservation of antigenic/control peptides in various species. When antibody crossreactivity has actually been experimentally confirmed in various species, it will be mentioned in the appropriate data sheets.

"Neat Antisera or antisera" are the unpurified antiserum and it is suitable for ELISA and Western.
"Affinity pure" IgG may be more suitable for immunohistochemical (IHC) applications and to reduce background in most immunological applications including ELISA and Western.
"Control peptides" can not be used for Western as they are very short peptides. They are intended for ELISA or antibody blocking studies to establish antibody specificity.
Western blot +ve protein controls, where available, are semi-pure, pure or recombinant proteins that are formulated in SDS-PAGE sample buffer. They are recommended to be used for Western (load 10 ul/lane) for visualization with antibodies.

All Products are for in vitro research use only.