Giang Nguyen
Biology 303
8 November 2007
Protein Tyrosine Phosphatase 1B Deficiency and Its Effects
Protein tyrosine phosphatase 1B (PTP1B) is an important enzyme involved in cellular signaling. In general, phosphatases are enzymes that remove a phosphate group from (i.e. dephosphorylate) their substrates, as opposed to protein kinases, another class of enzymes that add, rather than remove, a phosphate group. The activity or function of some proteins differs depending on whether they are in their phosphorylated or dephosphorylated state. Therefore, protein kinases and phosphatases can modulate the function of numerous proteins and, thus, affect cellular signaling. As its name suggests, protein tyrosine phosphatase IB dephosphorylates the tyrosine (an aromatic amino acid with a hydroxyl group, which is often the site of phosphorylation) residue of its substrate (11).
The several studies to be discussed in this paper were particularly interested in the role of or lack thereof PTP1B in the insulin signaling pathway and the Ras-MAPK and PI3K-Akt pathways, which in turn could affect, but were not limited to, glucose homeostasis and tumorigenesis (tumor formation), respectively (2, 4, 8, 10). PTP1B is known to be a negative regulator of the insulin pathway, part of the mechanism for maintaining glucose homeostasis (10). Insulin is released when there is a high level of glucose in the blood stream; it then binds to an insulin receptor, stimulating the transphosphorylation of the receptor’s tyrosine residues, which allows the receptor to better phosphorylate its target proteins (IRSs). PI3K is activated when it binds to IRSs, and its products can then activate Akt, eventually leading to an increased number of GLUT 4 proteins that are translocated to the plasma membrane (4). GLUT 4 is a passive transporter that transports glucose into cells, particularly, muscle and adipose (fat) cells. A greater number of GLUT 4 proteins in the membrane means that more glucose can be taken up by the cells, which helps restore high blood glucose level to normal level and, thus, maintains glucose homeostasis (4). PTP1B negatively regulates this pathway by acting on the insulin receptor, IRS-1, and IRS-2 by dephosphorylating and thereby inactivating them (10).
Insulin resistance, which is a cell’s reduced responsiveness to the action of insulin, is a characteristic of type 2 diabetes (4, 10). Because of its negative regulatory role in the insulin signaling cascade, PTP1B is of interest to many studies delving into its possible therapeutic use for type 2 diabetes. (8) Many previous studies have established a correlation between the overexpression of PTP1B and insulin resistance in muscle and adipose tissues (10). In contrast, a study lead by the Nieto-Vazquez et al. investigated insulin sensitivity in PTP1B-deficient myocytes under normal physiological and TNF-a-induced insulin resistant conditions (10). TNF-a is an adipokine, a type of protein secreted from adipose tissue that can alter glucose homeostasis and insulin sensitivity (10). Exposure to TNF-a has been reported to result in insulin resistance in adipocytes and myocytes (7, 9). Nieto-Vazquez et al. used immortalized myocytes (single muscle cells) from the skeletal muscle of wild-type mice and PTP1B-deficient mice. The immortalized myocytes maintained the phenotypic traits of skeletal muscle and had unaltered expression of the proteins involved in insulin signaling; however, these cells had lower GLUT4 and higher GLUT1 levels compared with primary cells (10).
Insulin receptor autophosphorylation stimulated by insulin in PTP1B-deficient myocytes was detected at a lower concentration of insulin, achieved maximal effect quicker, and had a twofold increase compared to wild-type myocytes. Also under insulin stimulation, PI3-kinase activity (recall that this is associated with phosphorylated insulin receptor, IRS-1 and IRS-2) had quicker activation and doubled maximal effect in the PTP1B-deficient myocytes compared to wild type myocytes. Similar results were also obtained for the insulin-induced phosphorylation of Akt. These results illustrated greater insulin sensitivity in PTP1B-deficient myocytes. Furthermore, PTP1B-deficient myocytes showed increased insulin-stimulated glucose uptake of up to 29% from the basal level and a slightly higher level of GLUT4 in the plasma membrane, which makes sense since GLUT4 is responsible for glucose uptake. Treatment of the myocytes with TNF-a, which is known to cause insulin resistance, yielded results suggesting that the lack of PTP1B protected myocytes against TNF-a-induced insulin resistance. PTP1B-deficient myocytes were unaffected by TNF-a in terms of glucose uptake and activation of Akt. Experiments done in vivo also found that glucose uptake and insulin-induced Akt phosphorylation were not affected by TNF-a in PTP1B-deficient mice. (8)
In another study, Delibegovic et al. (4) also looked at the influence of PTP1B deficiency in glucose homeostasis in mice. However, their study focused on clarifying the extent that PTP1B deficiency really plays in glucose homeostasis since the global deletion of PTP1B also affects body weight/adiposity, which, in turn, influences insulin sensitivity (4). The team was able to circumvent this problem by generating mice with muscle-specific deletion of PTP1B. These mice were feed either a high fat diet (HFD) or a normal chow diet. And compared to the control mice, they had similar body masses. Thus, muscle-specific deletion of PTP1B did not affect body weight/fat content and, consequently, could be used to assess the role of PTP1B deficiency (in muscle) in glucose homeostasis (4).
Muscle-specific-PTP1B-deficient mice had lower glucose levels compared to wild-type mice, and they exhibited a better ability to clear glucose even though the insulin level in both the control and the experimental groups remained the same. These results suggested that the deficient mice had a greater insulin sensitivity than that of the wild type mice. As in the other study, results from Delibegovic et al. also showed that basal glucose level in blood was lower (i.e. greater glucose uptake in muscle), and that glycogen synthesis (serving as glucose storage) increased in PTP1B-deficient mice. However glucose uptake by WAT and hepatic glucose production were not affected since the PTP1B deficiency was muscle-specific. These researchers also found that the levels of phosphorylated insulin receptors and IRS-1’s were greater in the deficient mice compared to the controls. In the controls, there was a difference in the level of phosphorylated insulin receptors between the high-fat-fed and chow-fed mice, with the level in mice on HFD being lower. (4)
Although most of the results from the two studies were similar, Delibegovic et al. did not find basal activation of P13K or Akt. They suggested that other phosphatases may have compensatory actions (keep in mind that the pathway is interconnected with numerous other pathways and is a lot more complicated). In addition, no increase in insulin signaling was observed in other tissues. The team concluded that increased “insulin- stimulated glucose uptake into muscle alone is sufficient to improve” body-wide glucose homeostasis (4). However, further research on other types of tissues should be done to understand fully the effects of PTP1B deficiency and its role in glucose homeostasis. In addition, these researchers found that rosiglitazone, which is used in treatment of type 2 diabetes, further improved glucose homeostasis and insulin sensitivity in PTP1B-deficient mice. Thus, its effect was “additive” to PTP1B deficiency in muscle (4).
In another study, the role of PTP1B in mammary tumorigenesis was explored (8). While the role of PTP1B as a negative regulator of the insulin signaling cascade is well known, the role of PTP1B in oncogenesis has not always been so clear. In fact, different studies often produced conflicting results. For example, in an in vitro study using immortalized fibroblasts, PTP1B was observed to have positive effect on Ras signaling (proliferation) (6) while in another in vivo experiment, PTP1B deficiency led to an increase, rather then the expected decrease, in B cell lymphoma (lymphocyte-originating cancer) in p53-dependent tumorigenesis (5).
To define the role of PTP1B in ErbB2-induced mammary tumorigenesis, Julien et al. (8) employed both genetic and pharmacological approaches. ErbB2 is a proto-ongocene that can be activated by point mutations, deletions, or insertions in the region coding its transmembrane or extracellular domain. (8). ErbB2 is over expressed in about 25% of all breast cancers (4, 8), and ErbB2 over expression is correlated with over expression of PPT1B (12). In this study, transgenic NDL2 mice (which over express ErbB2 and frequently develop multiple mammary tumors) were crossed with PTP1B-deficient mice to obtain transgenic NDL2 mice that were regular transgenic, heterozygous-, or homozygous-deficient in PTP1B. The homozygous PTP1B-deficient mice had delayed onset of tumorigenesis, reduced number of tumors after onset, and a lower percentage with lung metastasis compared to regular transgenic mice. Heterozygous mice gave intermediate results. Also, the histopathology, which is tissue change characteristic of or accompanying a disease, of the mammary glands in the deficient and non-deficient mice differed. Development of malignant tumors occurred sooner and the tumors were bigger in the regular transgenic mice compared to the PTP1B-deficient mice, who had delayed progression from hyperplasia (unusual increase in mass but benign) to carcinoma (malignant) (8). In addition, the levels of activated ErbB2 (those phosphorylated) and ErbB3 receptors were found to be much higher in the regular mice compared to the PTP1B-deficient mice. This result was expected since ErbB2 is over expressed in mammary tumors and tumors in non-deficient mice developed sooner in the regular mice. Furthermore, heterodimerization of the ErbB receptors, particularly the ErbB2-ErbB3 dimers, is essential to the ErbB receptor family’s role in “cell growth and transformation.” (3). Thus, an increase in the activity of one receptor would be expected to coincide with an increase in the other.
Ras-MAPK pathway and PI3K-Akt pathway are involved in cell proliferation and cell survival, respectively. (Keep in mind that in reality these pathways are much more complicated and are not as straightforward as will be presented here). In the Ras-MAPK pathway, it has been shown that increased level of PTP1B decreases the activity of p62dok (by dephosphorylating it), leading to increased Ras and p42/p44 MAPK activation (6). In the absence of PTP1B, the results were the opposite: there was more phosphorylated p62dok, less phosphorylated Ras, and less phosphorylated p44/p42MAPK, resulting in decreased Ras-MAPK signaling in PTP1B-deficient mice (8).
In tumors that overexpress ErbB2, the level of Akt, a serine/theronine kinase is highly activated (1). Similarly, a lower level of activated ErbB2 in PTP1B-deficient mice resulted in a lower level of phosphorylated Akt. Protein p27 and cyclin D1 are effectors in the PI3K-Akt pathway. The protein p27 arrests a cell in G1 phase by inhibiting cyclin D1. The level of p27 decreased over time for both non-deficient and deficient mice, while the level of cyclin D1 increased over time but was higher in non-deficient mice, consistent with tumor progression over time. Effectively, these data supported the idea that the PTP1B-deficiency in mice resulted in the down regulation of the PI3K-Akt pathway (8). In tumor cells over expressing ErbB2, hyperactivation of the PI3K-Akt pathway provides resistance to apoptosis (programmed cell death) (8). To determine the influence of PTP1B deficiency on apoptosis, cleavage analysis of poly (ADP-ribose) polymerase (PARP) and a determination of caspase-3 activation were carried out. Higher levels of cleaved PARP and caspase-3 (an enzyme activated when cleaved and is involved in apoptosis by cleaving other proteins) were found in PTP1B deficient mice, indicating more apoptosis events happening (8).Thus, a deficiency in PTP1B resulted in both decreased proliferation and increased cell death, which could partially explain the observed delayed onset and spread of mammary tumors in these mice compared to non-deficient mice. (8) Similar results were obtained by Bentires-Aji et al. (4) who showed that tumor formation was delayed in a different strain of PTP1B deficient mice that were monitored for 3 years (compared to approximately 1 year in the other study) (4). The delayed tumorigenesis only occurred with ErbB2-induced tumorigenesis, not with all types of breast cancer (4). Both studies suggested that PTP1B inhibitors could be used for selective therapy against ErbB2-induced tumors. However, further studies should be done to establish the effects of PTPIB inhibitors on other processes linked to cellular signaling.
In conclusion, a deficiency of PTP1B results in a multitude of effects because of the complexity of cellular signaling. Experiments have thus far shown that PTP1B deficiency delays ErbB2-induced tumorigenesis, protects against lung metastasis and TNF-a-induced insulin resistance, and improves glucose homeostasis. In addition, the inhibition of PTP1B may provide a means for therapeutic treatment for type 2 diabetes.
References
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