Conserved sequence domains in cell cycle regulatory proteins

Kay Hofmann and Philipp Bucher, Swiss Institute for Experimental Cancer Research, 1066 Epalinges, Switzerland.

Proteins involved in signal transduction and cell cycle regulation frequently consist of several independent domains, each of them mediating a particular type of interaction. Typically, such domains can be identified by analyzing regions of local sequence homology occurring in otherwise unrelated sequences. In some cases, like e.g. the SH2 or SH3 domain, these homologies are detectable by conventional sequence comparison methods. By application of 'generalized profiles', a very sensitive method for the detection of weak sequence similarities, we discovered several novel sequence motifs in proteins involved in cell cycle control. In collaboration with Amos Bairoch in Geneva, we are assembling a database of motif descriptors (PROSITE), which is made freely available and can be searched using free programs or WWW-servers.

Further information is available electronically from the following sources:

By E-mail from the authors (address es are given in the title page)
On the WWW, using the URL http://www.sib-isrec.ch for general informations, http://www.sib-isrec.ch/domains for information on conserved sequence domains, http://www.sib-isrec.ch/profile for information on generalized profiles (including an online version of this poster) and http://www.sib-isrec.ch/software for online searches of the PROSITE collection with a sequence.
The software necessary to run profile searches on a local computer is available by anonymous ftp from www.sib-isrec.ch in the directory pftools

The generalized sequence profile method

Application of sequence profiles is a very sensitive method for the discovery of distant sequence relationships. In contrast to conventional sequence comparison and database searching methods, not a single sequence is used as a query object but a profile constructed from a family of related sequences. These profiles are normally derived from multiple alignments of the initial sequence set. In addition to the sequences themselves, a profile contains the following information:

which types of residues are allowed at what position
which positions are important (=highly conserved), which ones are not
which positions or regions can allow insertions, which regions may be dispensable

For the detection of distant relationships, the assessment of statistical significance is very important. We derive an estimation of the error probability from an empirical score distribution obtained with a randomized protein database.

An additional advantage of the profile method is the possibility of iterative profile refinement. Sequences with highly significant similarity to the profile are aligned to the profile and included in the profile construction process for the next round of database searches.

Currently, we are constructing generalized profiles for the most important of the highly divergent protein domains and families. These profiles will be included in the PROSITE pattern collection. We are also trying to detect and describe previously unknown homology domains, with emphasis on proteins involved in signal transduction and regulatory pathways.

Selected references

The original profile method:

Gribskov, M., McLachlan, A.D. and Eisenberg,D. (1987), Proc. Natl. Acad. Sci. USA 84:4355-4358.

Improvements to the profile method:

Luthy, R., Xenarios, I. and Bucher, P. (1994), Prot. Sci. 3:139-146.

The generalized sequence profiles:

Bucher, P. and Bairoch A. (1994), in: Proceedings of the 2nd ISMB Conference, pp. 53-61, AAAI press.
Bucher, P., Karplus, K., Moeri, N. and Hofmann, K. (1996), Computers & Chemistry, in press.

The PROSITE pattern and profile library:

Bairoch, A., Bucher, P. and Hofmann, K. (1996), Nucleic Acids Res. 24:189-196.

The FHA-domain: A sequence motif possibly mediating phospho-Ser/Thr-specific interactions is found in several cell cycle regulatory proteins

The FHA-domain is a sequence motif found in several nuclear protein kinases, transcription factors, and other proteins. Several of these proteins are involved in cell cycle regulation, among them are :

S. pombe dma1, a protein involved in the spindle checkpoint (see poster B27)
S. cerevisiae DUN1 and RAD53/SAD1/MEC2/SPK1, two protein kinases linking the S-phase checkpoint to DNA-damage repair.
S. pombe cds1, a kinase acting in the S-phase checkpoint.

In the plant phosphatase KAPP, a region containing the FHA-domain in its center has been shown to interact specifically with a receptor-type Ser/Thr kinase only if the kinase is autophosphorylated. A multiple alignment of representative sequences and an updated list of proteins containing FHA-domains are shown below.

Hofmann, K. and Bucher, P. (1995), Trends Biochem. Sci. 20:347-349

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Abbreviations: FHA: FHA-domain; RF: RING-finger; FH: fork head domain; ZF: zinc finger; ATP,da: Walker1/2 ATP binding motifs; white vertical bars: predicted transmembrane regions.

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The UBA-domain: A sequence motif found in multiple enzyme classes of the ubiquitination pathway

The UBA-domain is a novel sequence motif found in several proteins having connections to ubiquitin and the ubiquitination pathway:

Bovine E2-25K and several other ubiquitin-conjugating enzymes (E2), catalyzing the second step in protein ubiquitination.
Drosophila hyperplastic discs protein, a putative ubiquitin-protein ligase (E3), catalyzing the third and final step in protein ubiquitination.
Human ubiquitin isopeptidase T and several other ubiquitin C-terminal hydrolases, catalyzing regulatory protein-deubiquitination.
S.cerevisiae RAD23 and its mammalian homologues. These proteins act in UV excision repair and contain a N-terminal domain similar to ubiquitin itself.
Mammalian proto-oncogene c-cbl, a protein interacting with multiple signal transduction factors. Cbl has recently been shown to undergo regulatory ubiquination upon macrophage stimulation.

A multiple alignment of representative sequences and the domain structure of proteins containing UBA-domains are shown below.

[gif] [Postscript]
Abbreviations: U: UBA-domain; H1,H2: Deubiquitinase catalytic domains; UBC: UBC catalytic domain; ub: ubiquitin-homology; RF: RING-finger; pab: PABP C-terminus homology; KA1: KA1-domain; EH: EPS15-homology domain; HECT: Ubiquitin-ligase (E3) catalytic domain; UX: UX-domain.

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The GRK-domain: a putative G_a-interacting protein domain.

The GRK-domain is a novel sequence motif found in several proteins working in signalling by heterotrimeric G-proteins. Sequences containing single or multiple copies of the GRK domain include:

S. cerevisiae alpha-factor signaling regulator SST2, a protein probably acting by desensitizing the alpha-factor response.
C. elegans G-protein signaling regulator EGL-10, which also contains a region similar to G_g-subunits.
Human G_a-interacting protein GAIP.
Human G0/G1 switch regulatory protein 8.
Human B-cell activation protein BL34.
The complete family of characterized G-protein coupled receptor kinases. These proteins phosphorylate activated G-protein coupled receptors, like e.g. the beta-adrenergic receptor, thus desensitizing the response. Several of these kinases possess a C-terminal PH-domain that might interact with G_bg-subunits and targets the kinase to the membrane. The presence of a GRK-domain in the N-terminal receptor-recognition region suggests the participation of G_a-subunits in the recognition process.

The domain structure of proteins containing GRK-domains is shown below.

[gif] [Postscript]
Abbreviations: GRK: GRK-domain; PH: PH-domain; GPg: G-protein gamma subunit homology; black box: GRK-specific extension of the kinase domain.

The BB-domain is present in multiple proteins of the STE pathway and other signal transduction proteins

The BB-domain is a sequence motif initially detected in the two BEM1- interacting proteins BEB1 and BOB1. It also occurs in several other proteins probably involved in signal transduction, including:

S. pombe ste4 protein.
S. pombe byr2 protein kinase and its homolog from S.cerevisiae, STE11.
Drosophila polyhomeotic proximal protein PHP.
Drosophila BicC protein.
S.cerevisiae BEB1 and BOB1, two proteins involved in budding.
A non-receptor tyrosine kinase from Dictyostelium.
Human LAR-interacting protein. In this protein, the interaction with the tyrosine phosphatase LAR has been mapped to the region containing three adjacent copies of the BB-domain.
The complete Eph-family of tyrosine kinases.

The domain structure of proteins containing BB-domains is shown below.

[gif] [Postscript]
Abbreviations: BB: GRK-domain; PH: PH-domain; SH3: SH3-domain; KH: KH-domain; A: Ankyrin-repeat; DHR: DHR/PDZ-domain; white vertical bars: predicted transmembrane regions.

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