Akira Miyazato

SOCS-1/JAB/SSI-1 Can Bind to and Suppress Tec Protein-tyrosine Kinase

Tec is the prototype of a recently emerging subfamily among nonreceptor type protein-tyrosine kinases and is known to become tyrosine-phosphorylated and activated by a wide range of cytokine stimulations in hematopoietic cells. Although Tec was recently shown to be involved in the cytokine-driven activation mechanism of c-fos transcription, it is yet obscure how Tec relays the signals from cell surface receptors to the nucleus. To identify signaling molecules acting downstream of Tec, we have looked for Tec-interacting proteins (TIPs) by using the yeast two-hybrid system. Here we report the identification and characterization of a novel protein, TIP3, which has been simultaneously identified by other groups as SOCS-1, JAB, or SSI-1. TIP3 carries one Src homology 2 domain with a sequence similarity to that of CIS. In 293 cells, TIP3 associates with Tec and suppresses its kinase activity. Interestingly, TIP3 can also down-regulate the activity of Jak2 but not that of Lyn. We propose that SOCS-1/JAB/SSI-1/TIP3 is a novel type of negative regulator to a subset of protein-tyrosine kinases.

Tec protein-tyrosine kinase is an effector molecule of Lyn protein-tyrosine kinase.

The Tec family is a recently emerging subfamily among nonreceptor type protein‐tyrosine kinases (PTKs) consisting of Tec, Txk, Btk, Bmx, and Itk/Tsk/Emt. They have a long amino‐terminal unique region containing a pleckstrin homology domain and a Tec‐homology domain. We could previously show that, through the Tec‐homology domain, Tec is bound to Lyn kinase both in vitro and in vivo. Because Tec is coexpressed with Lyn in many hematopoietic cell types, it has been intriguing to investigate the biological role of the Tec‐Lyn association. Here we demonstrate that Lyn can phospho‐rylate tyrosine residues of the Tec protein, and thereby activate Tec in 3T3 fibroblasts. However, coexpression of Tec has little effect on the phospho‐tyrosine‐contents of Lyn. By using the in vitro kinase assay and the yeast system, we could prove that the Tec protein is a direct substrate of the Lyn kinase both in vitro and in vivo. From this evidence we conclude that Tec acts downstream of Lyn in intracellular signaling pathways. This is a novel case where one PTK is phosphorylated and regulated by an‐other.—Mano, H., Yamashita, Y., Miyazato, A., Mi‐ura, Y., Ozawa, K. Tec protein‐tyrosine kinase is an effector molecule of Lyn protein‐tyrosine kinase. FASEB J. 10, 637‐642 (1996)

Grb10/GrbIR as an in vivo substrate of Tec tyrosine kinase

Tec is a member of the recently emerging subfamily among nonreceptor protein‐tyrosine kinases (PTKs). Although many members of this family have been shown to be involved in a wide range of cytokine‐mediated signalling systems, the molecular mechanism by which they exert in vivo effects remains obscure. To gain insights into the downstream pathways of Tec, we here looked for Tec‐interacting proteins (TIPs) by using the yeast two‐hybrid screening.

Severe hepatitis and complete molecular response caused by imatinib mesylate: Possible association of its serum concentration with clinical outcomes

A 40-year-old female with chronic myelogeneous leukemia (CML) in the chronic phase was treated with imatinib mesylate (STI571) because of interferon resistance. She achieved complete cytogenetic response but not complete molecular response 3 months after STI571 administration. Six months later, she developed severe liver damage without evidence of actively infectious hepatitis A, B, C, G, E, TT virus, Epstein-Barr virus or cytomegalovirus. A significant serum level of STI571 (107 ng/ml) was detected, although she had not taken the drug for 6 days. Liver biopsy demonstrated massive hepatic necrosis, consistent with drug-induced hepatitis. She achieved complete molecular response, although she did not take STI571 for 47 days after the development of hepatitis. These results suggest that both hepatitis and molecular response were associated with the serum STI571 concentration.

Regulation of Cellular Gene Expression in Endothelial Cells by Sin Nombre and Prospect Hill Viruses

Mechanisms of hantavirus-induced vascular leakage remain unknown. This study was initiated to determine whether hantavirus-induced changes in endothelial cell gene expression may provide insight into disease mechanisms. Additionally, by using pathogenic Sin Nombre virus (SNV) and non-pathogenic Prospect Hill virus (PHV), we wanted to identify cellular responses that are likely to differentiate pathogenic from nonpathogenic hantaviruses. Using the Affymetrix DNA Array, we found that PHV and SNV did not significantly differ in the number of activated genes (18 versus 14 genes) in infected endothelial cells at 4 h PI. However, a smaller group of genes (36) were up-regulated by PHV compared to SNV (175) at 12 h PI. Only two genes were down-regulated in SNV-infected cells. Expression of the functionally diverse group of genes was altered at an early stage of infection (4 and 12 h PI). The genes affected include putative anti-viral factors, transcription factors, growth factors, chemokines, receptors, structural proteins, metabolism, and kinases. Although many genes were activated in cells infected with SNV and PHV, overall cellular transcriptional responses were more altered by pathogenic SNV compared to non-pathogenic PHV.

The relationship of aplastic anemia and PNH

Bone marrow failure has been regarded as one of the triad of clinical manifestations of paroxysmal nocturnal hemoglobinuria (PNH), and PNH in turn has been described as a late clonal disease evolving in patients recovering from aplastic anemia. Better understanding of the pathophysiology of both diseases and improved tests for cell surface glycosylphosphatidylinositol (GPI)-linked proteins has radically altered this view. Flow cytometry of granulocytes shows evidence of an expanded PNH clone in a large proportion of marrow failure patients at the time of presentation: in our large NIH series, about 1/3 of over 200 aplastic anemia cases and almost 20% of more than 100 myelodysplasia cases. Clonal PNH expansion (rather than bone marrow failure) is strongly linked to the histocompatability antigen HLA.-DR2 in all clinical varieties of the disease, suggesting an immune component to its pathophysiology. An extrinsic mechanism of clonal expansion is also more consistent with knock-out mouse models and culture experiments with primary cells and cell lines, which have failed to demonstrate an intrinsic proliferative advantage for PNH cells. DNA chip analysis of multiple paired normal and PIG-A mutant cell lines and lymphoblastoid cells do not show any consistent differences in levels of gene expression. In aplastic anemia/PNH there is surprisingly limited utilization of the V-beta chain of the T cell receptor, and patients’ dominant T cell clones, which are functionally inhibitory of autologous hematopoiesis, use identical CDR3 regions for antigen binding. Phenotypically normal cells from PNH patients proliferate more poorly in culture than do the same patient’s PNH cells, and the normal cells are damaged as a result of apoptosis and overexpress Fas. Differences in protein degradation might play a dual role in pathophysiology, as GPI-linked proteins lacking an anchor would be predicted to be processed by the proteasome machinery and displayed in a class I H.A. context, in contrast to the normal pathway of cell surface membrane recycling, lysosomal degradation, and presentation by class II HLA. The strong relationship between a chronic, organ-specific immune destructive process and the expansion of a single mutant stem cell clone remains frustratingly enigmatic but likely to be the result of interesting biologic processes, with mechanisms that potentially cna be extended to the role of inflammation in producing premalignant syndromes.

Sak serine-threonine kinase acts as an effector of Tec tyrosine kinase.

The murine sak gene encodes a putative serine-threonine kinase which is homologous to the members of the Plk/Polo family. Although Sak protein is presumed to be involved in cell growth mechanism, efforts have failed to demonstrate its kinase activity. Little has been, therefore, elucidated how Sak is regulated and how Sak contributes to cell proliferation. Tec is a cytoplasmic protein-tyrosine kinase (PTK) which becomes activated by the stimulation of cytokine receptors, lymphocyte surface antigens, heterotrimeric G protein-linked receptors, and integrins. To clarify the in vivo function of Tec, we have tried to isolate the second messengers of Tec by using the yeast two-hybrid screening. One of such Tec-binding proteins turned out to be Sak. In human kidney 293 cells, Sak became tyrosine-phosphorylated by Tec, and the serine-threonine kinase activity of Sak was detected only under the presence of Tec, suggesting Sak to be an effector molecule of Tec. In addition, Tec activity efficiently protects Sak from the “PEST” sequence-dependent proteolysis. Internal deletion of the PEST sequences led to the stabilization of Sak proteins, and expression of these mutants acted suppressive to cell growth. Our data collectively supports a novel role of Sak acting in the PTK-mediated signaling pathway.

Mediation by the Protein-tyrosine Kinase Tec of Signaling between the B Cell Antigen Receptor and Dok-1

A variety of growth factor receptors induce the tyrosine phosphorylation of a nonreceptor protein-tyrosine kinase Tec as well as that of a Tec-binding protein of 62 kDa. Given the similarity in properties between this 62-kDa protein and p62Dok-1, the possibility that these two proteins are identical was investigated. Overexpression of a constitutively active form of Tec in a pro-B cell line induced the hyperphosphorylation of endogenous Dok-1. Tec also associated with Dok-1 in a phosphorylation-dependent manner in 293 cells. Tec mediated marked phosphorylation of Dok-1 both in vivo and in vitro, and this effect required both the Tec homology and Src homology 2 domains of Tec in addition to its kinase activity. Expression of Dok-1 in 293 cells induced inhibition of Ras activity, suggesting that Dok-1 is a negative regulator of Ras. In the immature B cell line Ramos, cross-linking of the B cell antigen receptor (BCR) resulted in tyrosine phosphorylation of Dok-1, and this effect was markedly inhibited by expression of dominant negative mutants of Tec. Furthermore, overexpression of Dok-1 inhibited activation of the c-fos promoter induced by stimulation of the BCR. These results suggest that Tec is an important mediator of signaling from the BCR to Dok-1.

Deletion of Src Homology 3 Domain Results in Constitutive Activation of Tec Protein‐Tyrosine Kinase

Tec protein‐tyrosine kinase (PTK) is the prototype of a new subfamily of non‐receptor type PTKs, and is abundantly expressed in hematopoietic tissues. We have revealed that Tec is inducibly tyrosine‐phosphorylated and activated by stimulation with a wide range of cytokines. To get more insight into the signaling mechanism through Tec, we have generated a constitutively active form of Tec PTK. Deletion of the Src homology (SH) 3 domain gave rise to a hyperphosphorylated and activated Tec kinase (TecΔSH3). The activity of TecΔSH3 was confirmed in 293 cells, as well as in cytokine‐dependent hematopoietic cells (BA/F3). TecΔSH3 should be a useful tool to study the in vivo substrates of Tec PTK.

New human myelodysplastic cell line, TER‐3: G‐CSF specific downregulation of Ca2+/calmodulin‐dependent protein kinase IV

We have established a new hematopoietic cell line from a patient with myelodysplastic syndrome (MDS), which was refractory anemia with excess blasts (RAEB). This cell line, designated TER‐3, depends on several cytokines for long‐term survival and growth, and requires interleukin‐3 (IL‐3) for continuous growth. Cytochemical analysis revealed that TER‐3 cells are weakly dianisidine positive and nonspecific esterase positive, but peroxidase negative. The surface marker profile shows that the TER‐3 cells are strongly positive for myeloid, lymphoid, and megakaryocytic antigens such as CD15, CD19, and CD61, and negative for some common multilineage antigens such as CD13, CD33, and CD34. Thus, this cell line has a multilineage phenotype, suggesting that the transformation event occurred in multipotent stem cells. Dianisidine‐ and nonspecific esterase‐positive TER‐3 cells increase with granulocyte‐colony stimulating factor (G‐CSF) rather than with IL‐3. These results suggest that the cell line is useful for understanding the mechanism underlying G‐CSF‐associated hematopoietic cell differentiation and activation in the patient with MDS. J. Cell. Physiol. 191: 183–190, 2002.