Sunday, July 13, 2008

src family kinases

In 1966, Francis Peyton Rous received the Nobel prize for Physiology or Medicine. He proposed that viruses can cause cancer and proved it in 1911. It was only because he lived almost forever that he lived to win the prize some 55 years after his scientific discovery. The delay can perhaps be attributed to the Nobel Committee awarding the 1926 prize to Johannes Andreas Grib Fibiger who had discovered the fungus Spiroptera carcinoma which he mistakenly believed was the cause of cancer. They didn’t want to make a second mistake and for a generation the idea that infection could cause cancer was thought to be heretical. It is said that people don’t change their minds, its just that the people who hold the old ideas die off.

Rous, who died in 1970 at the age of 91, worked on chickens. Some chickens grow a tumor called a fibrosarcoma. Rous minced up these sarcomas, centrifuged them down to remove the solidsl, and injected the liquid that remained into chicks. The chicks also developed sarcomas. The liquid contained a virus, now called the Rous sarcoma virus (RSV – note this is nothing to do with Respiratory Syncitial Virus, which causes chest infections in children).

Later work by others showed that RSV was a retrovirus like HIV. Retroviruses that don’t cause cancer contain three genes, called gag, pol, and env, but cancer causing retroviruses may also contain a gene called v-src (viral-sarcoma). It was found that the v-src gene in RSV is required for the formation of the Rous Sarcoma and that the other genes do not take part in causing the cancer.

Src codes for a tyrosine kinase which is required to transmit signals from cell surface to the nucleus where functions like movement and proliferation are controlled. The v-src protein lacks the C-terminal inhibitory phosphorylation site (tyrosine-527) that most tyrosine kinases have, and is therefore always switched on and can’t be switched off.

In 1979, J. Michael Bishop and Harold E. Varmus discovered that normal chickens contain a gene that is structurally closely-related to v-src. The normal cellular gene was called c-src (cellular-src). This discovery led to their being awarded the Nobel prize in 1989. c-src which is only activated under certain circumstances where it is required (e.g. growth factor signaling). v-src is therefore an example of an oncogene whereas c-src is a proto-oncogene.

This discovery changed the current thinking about cancer from a model wherein cancer is caused by a foreign substance (a viral gene) to one where a gene that is normally present in the cell can cause cancer.

The Src family includes nine members: Src, Lck, Hck, Fyn, Blk, Lyn, Fgr, Yes, and Yrk.

Lck or leukocyte-specific protein tyrosine kinase is found in lymphocytes, most commonly in T cells. It associates with the bits the CD4 and CD8 co-receptors on repectively T helper cells and cytotoxic T cells that poke through the cell membrane into the cell so as to assist signaling from the T cell receptor (TCR) complex. When the T cell receptor is engaged by the specific antigen presented by MHC, Lck acts to phosphorylate the intracellular chains of the CD3 and zeta-chains of the TCR complex, allowing another cytoplasmic tyrosine kinase called ZAP-70 to bind to them. Lck then phosphorylates and activates ZAP-70, which in turn phosphorylates another molecule in the signaling cascade called LAT (short for Linker of Activated T cells). The tyrosine phosphorylation cascade started up by Lck produces a flow of calcium (Ca2+) ions and activation of important signaling cascades within the lymphocyte.

Hck or Hemopoietic cell kinase is a protein-tyrosine kinase expressed in hemopoietic cell types. It may help couple the Fc receptor to the activation of the respiratory burst. In addition, it may play a role in neutrophil migration and in the degranulation of neutrophils.

Fyn (Feline yes-related protein) was previous known as slk (src-like kinase) but there was confusion with another Slk (Ste20-like kinase). It was also previously known as syn (src and yes related protein). It is a membrane-associated non-receptor protein tyrosine kinase of approximately 59kDa, Fyn is expressed predominately in tissues of neuronal and hematopoietic origin. Neuronal Fyn and hematopoietic Fyn differ at the junction of the SH2 and kinase domains due to tissue specific alternative splicing. Fyn has been shown to be involved in B-cell and T-cell activation as well as keratinocyte differentiation. In T-cells, Fyn associates with the T-cell antigen receptor and Thy-1. Fyn activation in response to integrin ligation occurs through association of Fyn and integrins with caveolin-1. Activated Fyn binds directly to Shc and phosphorylates it resulting in recruitment of Grb2 and Sos culminating in ERK/MAPK activation. (Sorry about all those acronyms – I will get around to explaining them later.)

Blk or B-lymphoid kinase Blk is a Src family protein tyrosine kinase expressed in all stages of B cell development. Activation of B cells by various ligands is accompanied by activation of Blk. It has been suggested that Blk is involved in the control of B cell differentiation and proliferation. Blk transcripts have also been detected in human thymocytes, but not in mature T cells, implicating that Blk may play an important role in thymopoiesis. Blk function may be redundant, however, as mice that do not express Blk are not impaired with respect to B cell development and immune response.

Yes is nothing to do with Meg Ryan. The cellular oncogene c-Yes and its viral homologue v-Yes (the transforming gene of Yamaguchi 73 and Esh avian sarcoma viruses) encode a 60 kilodalton, cytoplasmic, membrane-associated, protein-tyrosine kinase. Yes is ubiquitously expressed in many tissues and cells. Like other Src family members, Yes contains several conserved functional domains such as an N-terminal myristoylation (look it up in Wikipedia) sequence for membrane targeting, SH2 and SH3 domains, a kinase domain and a C-terminal, non-catalytic domain. There is also a growing body of evidence to indicate specificity in Yes signaling. Yes is activated downstream of a multitude of cell surface receptors, including receptor tyrosine kinases, G-protein-coupled receptors and cytokine receptors. Additionally, both Yes and Src kinases are activated during the cell cycle transition from G2 to M phase. Dysfunction of Yes is associated with the development of various cancers.

Yrk of course stands for Yes related kinase. While screening a chicken kidney cDNA library for the normal homolog of the yes oncogene, a clone that encoded a novel non-receptor type protein tyrosine kinase of the Src family was isolated. This gene product was named Yrk (York), as an acronym for Yes-related kinase. As predicted from the cDNA sequence, the Yrk protein consists of 536 amino acids and has all the canonical features of a Src kinase. At the amino terminus it contains a myristylation signal, followed by a unique domain, SH3 and SH2 motifs, an ATP binding site, a kinase region and a carboxy-terminal sequence with a potential regulatory tyrosine at position 530. The sequence of the Yrk protein showed 79% identity with human Fyn and 72% identity with chicken Yes. The sequence data together with Southern and Northern blot analyses showed that the chicken yrk gene is distinct from the chicken fyn gene. Antibodies generated against the unique domain of the yrk protein expressed in bacteria precipitated a 60-kDa protein that was active in an immune complex kinase assay and was phosphorylated on tyrosine. Expression of the Yrk protein in adult chicken tissues was elevated in cerebellum and spleen. Relatively high levels of Yrk were also found in lung and skin.

Lyn is a V-yes-1 Yamaguchi sarcoma viral related oncogene homolog. It is mainly expressed in hematopoietic cells and in neural tissues. In various hematopoietic cells, Lyn has emerged as a key enzyme involved in the regulation of cell activation. In these cells, a small amount of Lyn is associated with cell surface receptor proteins, including the BCR, CD40 and CD19. Following engagement of the receptors, Lyn undergoes rapid phosphorylation and activation. Lyn activation triggers a cascade of signaling events mediated by Lyn phosphorylation of tyrosine residues within the immunoreceptor tyrosine-based activation motifs (ITAM) of the receptor proteins, and subsequent recruitment and activation of other kinases including Syk, phosholipase Cγ2 (PLCγ2) and phosphatidyl inositol-3 kinase. These kinases provide activation signals, which play critical roles in proliferation, Ca2+ mobilization and cell differentiation.

Intriguingly, Lyn also plays an essential role in the transmission of inhibitory signals through phosphorylation of tyrosine residues within the immunoreceptor tyrosine-based inhibitory motifs (ITIM) in regulatory proteins such as CD22, PIR-B and FCγRIIb1. Their ITIM phosphorylation subsequently leads to recruitment and activation of phosphatases such as SHIP-1 and SHP-1, which further downmodulate signaling pathways, attenuate cell activation and can mediate tolerance. By acting as a key regulator of both positive and negative signals, Lyn plays an important role in B cells. In these, Lyn sets the threshold of cell signaling and maintains the balance between activation and inhibition. Lyn thus functions as a rheostat that modulates signaling rather than as a binary on-off switch.

FGR comes from the Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog. The encoded protein contains N-terminal sites for myristylation and palmitylation, a PTK domain, and SH2 and SH3 domains which are involved in mediating protein-protein interactions with phosphotyrosine-containing and proline-rich motifs, respectively. The protein localizes to plasma membrane ruffles, and functions as a negative regulator of cell migration and adhesion triggered by the beta-2 integrin signal transduction pathway. Infection with Epstein-Barr virus results in the overexpression of this gene. Multiple alternatively spliced variants, encoding the same protein, have been identified.

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