Kinase Family BCR

Kinase Classification: Group SAPPK: Family BCR

BCR is a putative atypical protein kinase which is fused to the Abl tyrosine kinase in most cases of chronic myelogenous leukemia. BCR does not have a typical kinase domain, and though it shows biochemical kinase activity, this may still be due to a co-purifying kinase.

Domain Structure

BCR is a multi-domain protein. The first exon (in vertebrates) encodes an oligomerization domain that also contains the kinase catalytic activity. This is followed by a RhoGEF domain, a PH-like domain, a C2 domain and a RhoGAP domain, implicating it in Rho small GTPase activation and inactivation, as well as membrane association. The structure of the oligomerization domain has been solved [1]. This comprises the first 72 AA of the 428 AA first exon in human. It has a largely helical structure and its oligomerization ability has been modeled. This is followed in exon 1 by a poorly conserved linked and a second region that is conserved in vertebrate BCR, but not well conserved in other paralogs or invertebrate orthologs.

Evolution

Homologs of human BCR can be found in all animals other than nematodes. The exon1 domain has been reported to be specific to vertebrate BCR, but can be seen to be weakly conserved in several other homologs, including insect RhoGAP1A and homologs as divergent as Hydra. ABR is a vertebrate paralog of BCR, whose published sequences lack the first exon. However, our analysis of ESTs and genomic sequences from throughout the vertebrates shows that ABR can be extended, with a single conserved upstream exon also encoding similarity to BCR exon 1. Another BCR-like gene has been found in fish, but not yet annotated. We call this ABRf for now. ABRf does not appear to have the exon1 domain. Fish also have a duplication of the BCR gene.

Multiple homologs also exist in some invertebrates. Drosophila RhoGAP1A includes homology to BCR exon 1, while RhoGAP100F has no recognizable similarity in this region.

An alignment of vertebrate BCR, ABR and ABRf, along with invertebrate BCR genes and the outgroup RhoGAP100F is at http://kinase.com/wikifiles/bcr.aln. This includes the extended ABR sequences with the BCR-exon 1 homology, and a variety of other novel gene predictions.

Biochemical Function

Kinase activity was first associated with BCR by immunoprecipitation [2]. An extended analysis was done to tell if the kinase activity was part of the BCR protein or from a copurifying kinase [3]. BCR was overexpressed in a bacculovirus expression system and purified by physicochemical means as well as by immunoprecipitation. Kinase activity was associated with a single (probably tetrameric) band by silver stain. In vitro serine autophosphorylation activity was about as active as for Abl (~0.5 pmol phosphate per pmol BCR in 30 minutes). Immunopurified BCR could also phosphorylate histones and casein on serine. Denaturated BCR was electrophoresed, blotted and renatured, and shown to retain autokinase activity. Deletion mutations mapped the kinase activity to the first 414 AA, within the first exon (the next smallest deletion, retaining AA 1-243 lacked kinase activity). Mutation of Cys-332 also abolished kinase activity. Cys-332 is conserved in vertebrate BCR and ABR, but not in invertebrate homologs.

Exon 1 also binds the SH2 domain of the Abl tyrosine kinase [4]. A number of subsequent paper have shown kinase activity associated with BCR immunoprecipitated from mammalian cells, but the kinase activity has not been further validated or dissected:

BCR immunoprecipitate has been also reported to bind to and phosphorylate AF-6 (MLLT4) [5]. No activity was seen when the first 167 AA of BCR was deleted. Immunopreciptation antibodies were rabbit polyclonal anti-Bcr C20 and anti-Bcr N20 antibody from Santa Cruz Biotechnology, but it is not clear what Ab was used for what experiment, though competition with the immunizing peptide blocked pull down of the kinase activity. BCR immunopreciptates were also reported to phosphorylate PPARg, also using IP with an undescribed antibody from Santa Cruz Biotechnology [6], and phosphorylated 14-3-3 Tau, when a GST-BCR fusion was purified with Glutathione beads [7].

Mutation of either Y328 or Y360, both likely phosphorylation targets of BCR-Abl, abolished transphosporylation activity of BCR, while the double mutant also blocked autophosphorylation [8]. This work used an antibody against anti-Bcr(1256 - 1271).

Function

The biological functions of BCR are poorly understood. The chief interest has been its role in leukemias: A reciprocal translocation between chromosmes 9 and 22 ('Philadelphia chromosome') is found in most cases of chronic myelogenous leukemia. It creates a fusion protein containing at least the first exon of BCR and the C-terminal regions of Abl, resulting in constitutive activation of the Abl tyrosine kinase activity. Oncogenic BCR fusions to the tyrosine kinases Jak2 and PDGFA have also been reported [9, 10], and BCR amplification associated with hepatocellular carcinoma [11]. The BCR-Abl fusion protein is inhibited by BCR itself, and residues shown to be required for kinase activity (Y260, S354) were also required for this inhibition [12]

References

1. Zhao X, Ghaffari S, Lodish H, Malashkevich VN, and Kim PS. Structure of the Bcr-Abl oncoprotein oligomerization domain. Nat Struct Biol. 2002 Feb;9(2):117-20. DOI:10.1038/nsb747 |
2. Stam K, Heisterkamp N, Reynolds FH Jr, and Groffen J. Evidence that the phl gene encodes a 160,000-dalton phosphoprotein with associated kinase activity. Mol Cell Biol. 1987 May;7(5):1955-60. DOI:10.1128/mcb.7.5.1955-1960.1987 |
3. Maru Y and Witte ON. The BCR gene encodes a novel serine/threonine kinase activity within a single exon. Cell. 1991 Nov 1;67(3):459-68. DOI:10.1016/0092-8674(91)90521-y |
4. Pendergast AM, Muller AJ, Havlik MH, Maru Y, and Witte ON. BCR sequences essential for transformation by the BCR-ABL oncogene bind to the ABL SH2 regulatory domain in a non-phosphotyrosine-dependent manner. Cell. 1991 Jul 12;66(1):161-71. DOI:10.1016/0092-8674(91)90148-r |
5. Radziwill G, Erdmann RA, Margelisch U, and Moelling K. The Bcr kinase downregulates Ras signaling by phosphorylating AF-6 and binding to its PDZ domain. Mol Cell Biol. 2003 Jul;23(13):4663-72. DOI:10.1128/MCB.23.13.4663-4672.2003 |
6. Alexis JD, Wang N, Che W, Lerner-Marmarosh N, Sahni A, Korshunov VA, Zou Y, Ding B, Yan C, Berk BC, and Abe J. Bcr kinase activation by angiotensin II inhibits peroxisome-proliferator-activated receptor gamma transcriptional activity in vascular smooth muscle cells. Circ Res. 2009 Jan 2;104(1):69-78. DOI:10.1161/CIRCRESAHA.108.188409 |
7. Clokie SJ, Cheung KY, Mackie S, Marquez R, Peden AH, and Aitken A. BCR kinase phosphorylates 14-3-3 Tau on residue 233. FEBS J. 2005 Aug;272(15):3767-76. DOI:10.1111/j.1742-4658.2005.04765.x |
8. Wu Y, Liu J, and Arlinghaus RB. Requirement of two specific tyrosine residues for the catalytic activity of Bcr serine/threonine kinase. Oncogene. 1998 Jan 8;16(1):141-6. DOI:10.1038/sj.onc.1201524 |
9. Griesinger F, Hennig H, Hillmer F, Podleschny M, Steffens R, Pies A, Wörmann B, Haase D, and Bohlander SK. A BCR-JAK2 fusion gene as the result of a t(9;22)(p24;q11.2) translocation in a patient with a clinically typical chronic myeloid leukemia. Genes Chromosomes Cancer. 2005 Nov;44(3):329-33. DOI:10.1002/gcc.20235 |
10. Wang HY, Thorson JA, Broome HE, Rashidi HH, Curtin PT, and Dell'Aquila ML. t(4;22)(q12;q11.2) involving presumptive platelet-derived growth factor receptor A and break cluster region in a patient with mixed phenotype acute leukemia. Hum Pathol. 2011 Dec;42(12):2029-36. DOI:10.1016/j.humpath.2010.07.028 |
11. Miyazaki Y, Mitsuma T, Ichida T, Odazima H, Ishihara K, and Asakura H. Amplification of BCR protein associated with oncogenesis in human hepatocellular carcinoma. Dig Dis Sci. 1997 May;42(5):927-37. DOI:10.1023/a:1018864414582 |
12. Perazzona B, Lin H, Sun T, Wang Y, and Arlinghaus R. Kinase domain mutants of Bcr enhance Bcr-Abl oncogenic effects. Oncogene. 2008 Apr 3;27(15):2208-14. DOI:10.1038/sj.onc.1210851 |