PTP1B (protein-tyrosine phosphatase 1B) is a non-transmembrane enzyme that is found on the endoplasmic reticulum (ER). Its significance stems from being a negative regulator of insulin as well as leptin signaling. The PTP1B dephosporylates, or in other words removes a phosphate group from the insulin receptor, IR, and also its primary substrates, which are called the Insulin Receptor Substrate proteins (IRS proteins). PTP1B in leptin, removes the phosphate from the tyrosine kinase, JAK2, which is called Janus kinase 2. More recently, it has been found to be a contributing factor in the onset of tumors, and has been linked more directly with breast cancer. It is also considered a potential drug target as its inhibition may lead to a stop in type 2 diabetes, obesity, and some forms of cancer.
Structure of PTP1BEdit
PTP1B's structure composes of approximately 800 residues. The protein is made up of an N-terminal catalytic phophatase domain followed by a regulatory region and a membrane localization domain. This is attached to the endoplasmic retilum.
Inhibition of Insulin Receptors, and Correlations with DiabetesEdit
When PTP1B was first discovered, it was found to inhibit both the insulin and leptin receptors. PTP1B does this by de-phosphorylating the insulin receptor (IR) and its primary substrates such as the IRS proteins. In turn, this results in patients developing diabetes. Ptp1b knockout mice demonstrated persuasive evidence that inhibiting this enzyme will allow people to stay lean and energetic independent of what or how much they eat. When the enzyme was disabled in the mice, they became hypersensitive to insulin and were lean despite being on a high-fat diet. The specific tissue location of where to disable the enzyme did not seem to matter much and all experiments pointed to the same result of fit and energetic mice with improved insulin sensitivity and increased glucose tolerance.
PTP1B is an enzyme that is known for being expressed with great abundance. It consists of an N-Terminal catalytic phosphatase region, being anywhere from 1 to 300 residues long, a regulatory region that ranges from 80 to 100 residues, and finally, a membrane localization domain which ranges from 400 to 435 residues long. The membrane localization domain ties, or bonds the PTP1B enzyme to the cytoplasmic face of the endoplasmic reticulum. Expression of PTP1B and its catalytic activity is usually strictly controlled by four mechanisms which can sometimes work together: oxidation, phosphorylation, sumoylation, and proteolysis.
PTP1B can be regulated in vivo by both reversible and irreversible oxidation. Cys 215, an amino acid at one of its active sites is positioned in an unusually acidic environment, which subsequently deprotonates at physiological pH. This converts the amino acid into an excellent nucleophile in catalysis. Once this conversion is made, it leaves Cys 215 susceptible to being inactivated by other highly reactive species containing oxygen. Depending on which oxygen-containing highly reactive species is used to modify PTP1b, the enzyme is oxidized to different oxidation states.
Through the use of crystallographic analysis, results have shown that if hydrogen peroxide is used, the sulphenic acid form of the PTP1B would be converted into the inactivated cyclic sulphenamide state through the process of oxidation. As this process takes place, a conformational change also occurs at the active site, which as a result exposes the hidden tyrosine amino acid, which is located at the phosphotyrosine binding loop. This process is predicted to be a reversible process. In contrast, the process would be irreversible if the enzyme is oxidized to the sulphinic or sulphonic state.
Although phosphorylation of both serine and tyrosine occur at multiple locations, the effects of phosphorylation remain controversial. Studies of phosphorylation at different serine residues on the enzyme produces contradicting results. For example, the phosphorylation of S378 and S352 by protein kinase C during metaphase or in response to external stimuli such as osmotic pressure does not significantly change the enzyme's activity level. However, when S50 is phosphorylated by AKT, the enzyme shows a decrease in its ability to dephosphorylate insulin receptors. Interestingly, when the same S50 residue is phosphorylated by CDC-like kinase 1 and 2, the enzyme shows a two-fold increase in its phosphatase acitivity. In a similar manner, studies of phosphorlyation at the tyrosine sites (Y66, Y152, Y153) on the enzyme have shown that these changes to PTP1b can either increase or decrease the protein's activity.
Small ubiquitin-related modifier (SUMO) proteins have recently been found to be important regulators for many cell functions. SUMO conjugation significantly regulates many protein characteristics e.g. stability, localization, interactions and activity. PTP1B is found to interact with a SUMO E3 ligase which encourages modification of PTP1B by SUMO. Enzymatic activity is reduced and therefore PTP1B is less active with substrates. The specific location of where SUMO modification takes place is still unclear. However, it has been observed that PTP1B accumulates in punctuate structures, which are located in the perinuclear region and the C-terminal. It is important to point out that the presence of the ER targeting domain of PTP1B is necessary for the maximal sumoylation to occur.
Calpain, a protease mediates the cleavage of the ER targeting part of PTP1B (C-terminal). This occurs in platelets that are activated and the result is an activated enzyme. Studies show that when calpain-1 in mice are disrupted, there is a lowered protein tyrosine phosphorylation level. When the platelets of these mice are analyzed, there was a large increase in the amount of PTP1B. This suggests that when the c-terminal is cleaved, PTP1B is cut into inactive pieces (fragments). Evidence that supports the proteolyzation, or the breakdown of protein PTP1B into inactive fragments is that the tyrosine phosphorylation defects, which are linked to the loss of calpain-1, were rescued in Capn1, along with the aggregation of the platelet.
It is important to note that other reports have shown that when PTP1B is reversibly oxidized, it would be inactivated by calpain-mediated cleavage in the catalytic domain. Thus, this led some to believe that whether the enzyme would be activated by cleavage of the C-terminus or inactivated by complete proteolysis (both processes mediated by the calpain protease) actually depends on PTP1B's oxidation state. However, no reports to date have reported the inactivation of the enzyme by calpain in any other cell types other than platelets.
Substrates of PTP1BEdit
Since PTP1B is located on the cytoplasmic face of the ER, the mechanisms that allow this enzyme to encounter and dephosphorylate its many different substrates were put into question. So far, four possible mechanisms are proposed. First, if the substrate is a membrane-bound receptor protein tyrosine kinase such as the insulin receptor, through a vesicle-mediated endocytosis process, these activated receptors would be internalized and brought into contact with the PTP1B enzyme. Second, evidences from bioluminescence resonance energy transfer-based and fluoresence resonance energy transfer-based live images support that PTP1B might be able to dephosphorylate the insulin receptor during its biosynthesis. Third, the part of the ER that PTP1B binds to is stretchable. This makes interaction between PTP1B and its substrates on the plasma membrane possible. Finally, adaptor proteins link PTP1B to its substrate, facilitating their interaction by forming a ternary complex. In addition to these mechanisms, the enzyme has binding motifs that facilitate its interaction with substrates such as the IR. Studies have shown that one of these motifs, the Y152 located in the β9-β10 turn of the catalytic domain, allows PTP1B to interact with the back side of the IR dimers.
Diabetes & ObesityEdit
Ptp1b knockout mice demonstrated persuasive evidence that inhibiting this enzyme will allow people to stay lean and energetic independent of what or how much they eat. When the enzyme was disabled in the mice, they became hypersensitive to insulin and were lean despite being on a high-fat diet. The specific tissue location of where to disable the enzyme did not seem to matter much and all experiments pointed to the same result of fit and energetic mice with improved insulin sensitivity and increased glucose tolerance.
So far, PTP1b has not been found to be a tumor suppressor but evidence suggests that it may be a negative regulator of cell growth. The enzyme can encourage cell death (apoptosis) and therefore potentially be a tumor suppressor. The inhibition of this enzyme may seem to solve many health problems very simply and some may wonder why action had not been taken sooner but as always, biology is not that simple. The total effects of the inhibition of PTP1B is still not known and some studies already suggest that inhibiting this enzyme will promote some forms of cancer. Some human cancers e.g. cancer of the breast and ovary show elevated levels of PTP1B. For breast cancer, activation of ErbB2 leads to higher levels of PTP1B expression. Studies using transgenic mice show that PTP1B is a positive regulator of the ErbB2 induced mammary tumorigenesis. The mice without PTP1B had a large delay of the onset of ErbB2 whereas the mice with an over-expression of PTP1B developed breast tumors. Using a PTP1B inhibitor in a mouse cancer model, Julien et al. 's results shown that this inhibitor effectively protected mice from the ErbB2 oncogene. However, even in PTP1B deficient models, breast tumors caused by the polyoma middle T antigen can still develop. Thus, PTP1B is proposed as an enzyme that plays a selective but contributing role in oncogenic signaling.
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