G. Wayne Zhou

The function of the protein tyrosine phosphatase SHP-1 in cancer

SHP-1, an SH2 domain-containing protein tyrosine phosphatase, is primarily expressed in hematopoietic cells and behaves as a key regulator controlling intracellular phosphotyrosine levels in lymphocytes. SHP-1 has been proposed as a candidate tumor suppressor gene in lymphoma, leukemia and other cancers, as it functions as an antagonist to the growth-promoting and oncogenic potentials of tyrosine kinase. The decreased levels of SHP-1 protein and SHP-1 mRNA observed in various leukemia and lymphoma cell lines have been attributed to either the methylation of the promoter region of the SHP-1 gene or the post-transcriptional block of SHP-1 protein synthesis. In contrast, SHP-1 protein is normally or over-expressed in some non-lymphocytic cell lines, such as prostate cancer, ovarian cancer and breast cancer cell lines. SHP-1 expression also is decreased in some breast cancer cell lines with negative expression of estrogen receptor as well as some prostate and colorectal cancer cell lines. These data suggest that SHP-1 can play either negative or positive roles in regulating signal transduction pathways. Dysfunction in SHP-1 regulation can cause abnormal cell growth and induce different kinds of cancers. In this paper, we summarize recent studies on the expression and regulation of SHP-1 protein and its pathological function in the development of lymphoma, leukemia and other cancers.

Crystal structure of the catalytic domain of protein-tyrosine phosphatase SHP-1

The crystal structures of the protein-tyrosine phosphatase SHP-1 catalytic domain and the complex it forms with the substrate analogue tungstate have been determined and refined to crystallographic R values of 0.209 at 2.5 Å resolution and 0.207 at 2.8 Å resolution, respectively. Despite low sequence similarity, the catalytic domain of SHP-1 shows high similarity in secondary and tertiary structures with other protein-tyrosine phosphatases (PTPs). In contrast to the conformational changes observed in the crystal structures of PTP1B and Yersinia PTP, the WPD loop (Trp419-Pro428) in the catalytic domain of SHP-1 moves away from the substrate binding pocket after binding the tungstate ion. Sequence alignment and structural analysis suggest that the residues in the WPD loop, especially the amino acid following Asp421, are critical for the movement of WPD loop on binding substrates and the specific activity of protein-tyrosine phosphatases. Our mutagenesis and kinetic measurements have supported this hypothesis.

Activation of the Akt-related cytokine-independent survival kinase requires interaction of its phox domain with endosomal phosphatidylinositol 3-phosphate

Protein kinases of the Akt and related serum- and glucocorticoid-regulated kinase (SGK) families are major downstream mediators of phosphatidylinositol (PI) 3-kinase signaling to many cellular processes including metabolic flux, membrane trafficking, and apoptosis. Activation of these kinases is thought to occur at the plasma membrane through their serine and threonine phosphorylation by the phosphoinositide-dependent kinase 1 (PDK1) protein kinase, which interacts with membrane 3′-polyphosphoinositides through its pleckstrin homology (PH) domain. Here, we demonstrate that the SGK family member cytokine-independent survival kinase (CISK) binds strongly and selectively to the monophosphoinositide PI(3)P through its phox homology (PX) domain. Comparing native green fluorescent protein-CISK (EGFP-CISK) to a mutant EGFP-CISK (Y51A) that displays attenuated binding to PI(3)P reveals that this interaction is both necessary and sufficient for its localization to early endosome antigen (EEA1)-positive endosomes. Furthermore, early endosome association of expressed epitope-tagged CISK in COS cells directed by binding of its PX domain to PI(3)P is required for activation of the CISK protein kinase by both insulin-like growth factor-1 and epidermal growth factor. Taken together, these results reveal a critical role of endosomal PI(3)P in the signal transmission mechanism whereby this survival kinase is activated in response to PI3-kinase stimulation by growth factors.

Crystal structure of the nuclear matrix targeting signal of the transcription factor acute myelogenous leukemia-1/polyoma enhancer-binding protein 2αB/core binding factor α2

Transcription factors of the acute myelogenous leukemia (AML)/polyoma enhancer-binding protein (PEBP2α)/core-binding factor α (CBFA) class are key transactivators of tissue-specific genes of the hematopoietic and bone lineages. AML-1/PEBP2αB/CBFA2 proteins participating in transcription are associated with the nuclear matrix. This association is solely dependent on a highly conserved C-terminal protein segment, designated the nuclear matrix targeting signal (NMTS). The NMTS of AML-1 is physically distinct from the nuclear localization signal, operates autonomously, and supports transactivation. Our data indicate that the related AML-3 and AML-2 proteins are also targeted to the nuclear matrix in situ by analogous C-terminal domains. Here we report the first crystal structure of an NMTS in an AML-1 segment fused to glutathione S-transferase. The model of the NMTS consists of two loops connected by a flexible U-shaped peptide chain.

Structural Basis for Substrate Specificity of Protein-tyrosine Phosphatase SHP-1

The substrate specificity of the catalytic domain of SHP-1, an important regulator in the proliferation and development of hematopoietic cells, is critical for understanding the physiological functions of SHP-1. Here we report the crystal structures of the catalytic domain of SHP-1 complexed with two peptide substrates derived from SIRPα, a member of the signal-regulatory proteins. We show that the variable β5-loop-β6 motif confers SHP-1 substrate specificity at the P-4 and further N-terminal subpockets. We also observe a novel residue shift at P-2, the highly conserved subpocket in protein- tyrosine phosphatases. Our observations provide new insight into the substrate specificity of SHP-1.

SHP‐1 suppresses cancer cell growth by promoting degradation of JAK kinases

SHP‐1 has been proposed to be a tumor suppressor gene for several cancers. The expression of SHP‐1 protein is diminished or abolished in most leukemia and lymphoma cell lines and tissues, and in some non‐hematopoietic cancer cell lines, such as estrogen receptor (ER) negative breast cancer cell lines and some colorectal cancer cell lines. However, we do not know whether the reduced SHP‐1 expression is the cause of cancer diseases or the secondary effect of cancer developments. Here, we first demonstrate that SHP‐1 has general tumor suppressing function in SHP‐1 transfected cell lines. Transfected SHP‐1 inhibits the growth of three lymphoma/leukemia cell lines (Ramos, H9, Jurkat) and one breast cancer cell line (HTB26). We also demonstrate a possible molecular mechanism for the tumor suppressing function of SHP‐1: SHP‐1 inhibits cell growth partly by negative regulation of activated JAK kinase. In addition, we find, for the first time, that SHP‐1 down‐regulates the level of TYK2 kinase in H9 cells and of JAK1 kinase in HTB26 cells, by accelerating their degradation. The SHP‐1 accelerated degradation of JAK1 kinase in HTB26 cells was blocked with the treatment of MG132, a specific inhibitor for proteasome‐mediated proteolysis. Our data suggest a new function of SHP‐1 in the regulation of proteasome‐mediated degradation pathway.

Structural analysis of regulatory protein domains using GST-fusion proteins

The glutathione S-transferase (GST) fusion protein expression system has been used extensively to generate a large quantity of proteins for structural studies. To avoid the inter-domain flexibility introduced by the GST segment, GST-fusion proteins are normally cleaved with proteases to release the GST moiety prior to crystallization. Recently, several reports have shown that GST-fusion proteins can also be used as a vehicle to determine the crystal structures of the attached small peptides and biological regulatory domains. In comparison with the standard method, GST-fusion proteins are more easily crystallized under similar conditions. In addition, the structure of the desired protein or peptide can be determined using the molecular replacement method with the help of the GST structure. Thus, GST-fusion proteins can be used as a new technique for structural determination of small regulatory domains, especially of small peptides. Here, we review the recent progress on this technique, known as GST-driven crystallization. We have summarized and compared different methods of protein preparation and crystallization used by different groups. We have also compared the three-dimensional structures, especially those of the fused peptide segments. Finally, we have discussed the potential effects of the crystal packing on the crystal structure.

Crystal structure of human protein tyrosine phosphatase SHP‐1 in the open conformation

SHP‐1 belongs to the family of non‐receptor protein tyrosine phosphatases (PTPs) and generally acts as a negative regulator in a variety of cellular signaling pathways. Previously, the crystal structures of the tail‐truncated SHP‐1 and SHP‐2 revealed an autoinhibitory conformation. To understand the regulatory mechanism of SHP‐1, we have determined the crystal structure of the full‐length SHP‐1 at 3.1 Å. Although the tail was disordered in current structure, the huge conformational rearrangement of the N‐SH2 domain and the incorporation of sulfate ions into the ligand‐binding site of each domain indicate that the SHP‐1 is in the open conformation. The N‐SH2 domain in current structure is shifted away from the active site of the PTP domain to the other side of the C‐SH2 domain, resulting in exposure of the active site. Meanwhile, the C‐SH2 domain is twisted anticlockwise by about 110°. In addition, a set of new interactions between two SH2 domains and between the N‐SH2 and the catalytic domains is identified, which could be responsible for the stabilization of SHP‐1 in the open conformation. Based on the structural comparison, a model for the activation of SHP‐1 is proposed. J. Cell. Biochem. 112: 2062–2071, 2011.

p47phox PX domain of NADPH oxidase targets cell membrane via moesin‐mediated association with the actin cytoskeleton

Activation of phagocytic NADPH oxidase requires association of its cytosolic subunits with the membrane‐bound flavocytochrome. Extensive phosphorylation of the p47phox subunit of NADPH oxidase marks the initiation of this activation process. The p47phox subunit then translocates to the plasma membrane, bringing the p67phox subunit to cytochrome b558 to form the active NADPH oxidase complex. However, the detailed mechanism for targeting the p47phox subunit to the cell membrane during activation still remains unclear. Here, we show that the p47phox PX domain is responsible for translocating the p47phox subunit to the plasma membrane for subsequent activation of NADPH oxidase. We also demonstrate that translocation of the p47phox PX domain to the plasma membrane is not due to interactions with phospholipids but rather to association with the actin cytoskeleton. This association is mediated by direct interaction between the p47phox PX domain and moesin.

Protein Expression Profiling of Breast Cancer Cells by Dissociable Antibody Microarray (DAMA) Staining

Dissociable antibody microarray (DAMA) staining is a technology that combines protein microarrays with traditional immunostaining techniques. It can simultaneously determine the expression and subcellular location of hundreds of proteins in cultured cells and tissue samples. We developed this technology and demonstrated its application in identifying potential biomarkers for breast cancer. We compared the expression profiles of 312 proteins among three normal breast cell lines and seven breast cancer cell lines and identified 10 differentially expressed proteins by the data analysis program DAMAPEP (DAMA protein expression profiling). Among those proteins, RAIDD, Rb p107, Rb p130, SRF, and Tyk2 were confirmed by Western blot and statistical analysis to have higher expression levels in breast cancer cells than in normal breast cells. These proteins could be potential biomarkers for the diagnosis of breast cancer.