Tomohiro Kurosaki

Reconstitution of Syk Function by the ZAP-70 Protein Tyrosine Kinase

ZAP-70 and Syk are PTKs required for TCR and BCR function, respectively. Loss of the Syk PTK results in a nonfunctional BCR. We provide evidence here that ZAP-70 and Syk are functionally homologous in antigen receptor signaling by demonstrating that expression of ZAP-70 in Syk- B cells reconstitutes BCR function. Reconstitution required the presence of functional Src homology 2 (SH2) and catalytic domains of ZAP-70. Thus, drug targeting of a single SH2 domain within ZAP-70 should be sufficient to inhibit hematopoietic antigen receptor function. In addition, we demonstrate that both ZAP-70 and Syk can bind directly to the phosphorylated Ig alpha and Ig beta subunits with affinities comparable to their binding to the TCR CD3 epsilon subunit. These data suggest that ZAP-70 and Syk are comparable in their abilities to mediate hematopoietic antigen receptor signaling.

Functional analysis of Csk in signal transduction through the B-cell antigen receptor.

In B cells, two classes of protein tyrosine kinases (PTKs), the Src family of PTKs (Lyn, Fyn, Lck, and Blk) and non-Src family of PTKs (Syk), are known to be involved in signal transduction induced by the stimulation of the B-cell antigen receptor (BCR). Previous studies using Lyn-negative chicken B-cell clones revealed that Lyn is necessary for transduction of signals through the BCR. The kinase activity of the Src family of PTKs is negatively regulated by phosphorylation at the C-terminal tyrosine residue, and the PTK Csk has been demonstrated to phosphorylate this C-terminal residue of the Src family of PTKs. To investigate the role of Csk in BCR signaling, Csk-negative chicken B-cell clones were generated. In these Csk-negative cells, Lyn became constitutively active and highly phosphorylated at the autophosphorylation site, indicating that Csk is necessary to sustain Lyn in an inactive state. Since the C-terminal tyrosine phosphorylation of Lyn is barely detectable in the unstimulated, wild-type B cells, our data suggest that the activities of Csk and a certain protein tyrosine phosphatase(s) are balanced to maintain Lyn at a hypophosphorylated and inactive state. Moreover, we show that the kinase activity of Syk was also constitutively activated in Csk-negative cells. The degree of activation of both the Lyn and Syk kinases in Csk-negative cells was comparable to that observed in wild-type cells after BCR stimulation. However, BCR stimulation was still necessary in Csk-negative cells to elicit tyrosine phosphorylation of cellular proteins, as well as calcium mobilization and inositol 1,4,5-trisphosphate generation. These results suggest that not only activation of the Lyn and Syk kinases but also additional signals induced by the cross-linking of the BCR are required for full transduction of BCR signaling.

Genetic Evidence for a Tyrosine Kinase Cascade Preceding the Mitogen-activated Protein Kinase Cascade in Vertebrate G Protein Signaling

The signal transduction pathway from heterotrimeric G proteins to the mitogen-activated protein kinase (MAPK) cascade is best understood in the yeast mating pheromone response, in which a serine/threonine protein kinase (STE20) serves as the critical linking component. Little is known in metazoans on how G proteins and the MAPK cascade are coupled. Here we provide genetic and biochemical evidence that a tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells. Targeted deletion of tyrosine kinase Csk in avian B lymphoma cells blocks the stimulation of MAPK by Gq-, but not Gi-, coupled receptors. In cells deficient in Bruton’s tyrosine kinase (Btk), Gi-coupled receptors failed to activate MAPK, while Gq-coupled receptor-mediated stimulation is unaffected. Taken together with our previous data on tyrosine kinases Lyn and Syk, the Gq-coupled pathway requires tyrosine kinases Csk, Lyn, and Syk, while the Gi-coupled pathway requires tyrosine kinases Btk and Syk to feed into the MAPK cascade in these cells. The central role of Syk is further strengthened by data showing that Syk can bind to purified Lyn, Csk, or Btk.

The domain structure and the function of poly(ADP-ribose) synthetase

Poly(ADP-ribose) synthetase is a chromatin-bound enzyme which synthesizes a protein-bound homopolymer of ADP-ribose utilizing NAD as a substrate. The characteristic nature of this enzyme is that it requires DNA for catalytic activity. The enzyme is rich in malignant tumor cells as well as in normal tissues where cell proliferation is very rapid. The enzyme has been purified to homogeneity from calf thymus, mouse testis and human placenta. The amino acid composition of these enzymes is very similar and a monoclonal antibody as well as antisera against the calf enzyme cross-reacts with mouse, chicken and human enzymes, suggesting that the antigenic structures of poly(ADP-ribose) synthetase are highly conserved in various animal cells. The native enzyme (Mr = 120K) is cleaved by limited proteolytic digestion into three different domains (Mr = 46K, 22K, 54K), the first containing the site for DNA binding, the second containing the site for automodification and the third containing the site for NAD binding. The DNA binding domain (Mr = 46K), like the native enzyme, has the ability to preferentially suppress nick induced random transcription initiation in a HeLa cell lysate, resulting in the production of run-off RNA initiated from the correct late promoter site on truncated DNA of adenovirus 2. The native enzyme poly(ADP-ribosyl)ates RNA polymerase and some other nuclear enzymes. These results, taken together, indicate that poly(ADP-ribose) synthetase plays a critical role in regulating gene expression in various eukaryotic cells.