The T-cell receptor utilizes two principal activation pathways, namely PKC and one or more tyrosine protein kinases (TPK's). Our overall objective is to show that these pathways cooperate and interact with accessory receptor signals to constitute a complex signalling network which regulates many T- lymphocyte functions. One approach will be to use a kinase component, which requires both threonine and tyrosine phosphorylation to exert its function as a microtubule associated protein-2 kinase (MAP-2K), to gain insight into this kinase cascade (aims 1-3). We are particularly interested to identify TPK's which may interact with 43 kDa lymphoid MAP-2K and will use in vitro reconstitution of purified kinases and cell lines which differ in their use of signalling pathways to perform these studies. We will study the relationship between threonine and tyrosine phosphorylation of MAP-2K as well as the possibility that PKC may be involved in either of these processes. These experiments will make use of kinase inhibitors, autophosphorylation reactions, phosphoamino acid analysis, and immune complex kinase assays. Additional control over these processes by accessory receptors such as CD4, which controls pp56lck activity, and the CD45 family, which expresses intrinsic tyrosine phosphatase activity, will be studied in memory and naive T-cells to develop an understanding of their activation pathways (aim 4). We are at the point of sequencing MAP-2K cDNA clones from a lymphoid library, and should be able to clarify lymphoid isoforms and their relationship to the rat ERK family. We will use these cDNA copies to prepare recombinant MAP- 2K protein and antibodies, and will study the molecular biology of MAP-2K in T-lymphocytes (aim 1). We would like to expand our observations which show that MAP-2K may play a role in G1 phase progression by doing anti- sense inhibition studies and will study activation of the kinase by transferrin and IL-2 receptors (aim 3). It is also possible that through the utility of a T-lymphocyte culture model for tolerance induction in mature cells, that MAP-2K activation and tyrosine phosphorylation of proteins may help to clarify a signal which synergizes with CD3 and prevent tolerization (aim 4). Closely aligned to our aim with MAP-2K is the study of a 63 kDa serine kinase which phosphorylates a consensus site on c-fos and is activated by CD3 and PMA. Although not a MAP-2K, fos-kinase may be related to 62.6 kDa ERK-3. We will study their possible homology by cloning the ERK-3 gene from a human and rat cDNA library with a view to producing a recombinant protein. This will be used to raise an antibody and as a source of kinase activity. We will determine whether c-fos is phosphorylated during TCR ligation and whether this modification influences nuclear localization and transcriptional autoregulation of c-fos. Our long-term goal in understanding T-cell activation is to achieve biological response modification by activators/inhibitors of signalling components.
NIH ExPorter
NIH ExPorter