SAM domains (also known as Pointed, SPM, and HLH domains) are frequently found in eukaryotic regulatory proteins ranging from receptor tyrosine kinases to transcription factors (1-3). Structures of several SAM domains reveal a common tertiary fold but show a diverse array of oligomeric states and binding schemes (4 -11). Some SAM domains, such as that from the Ets family transcription factor TEL, can self-associate to form an open-ended polymeric structure (10), whereas the closely related Ets-1, GABP␣, and Erg SAM domains are monomeric (4, 12, 13). The SAM domains from Eph receptor tyrosine kinases can either be monomeric or dimeric or may possibly form an extended oligomeric structure (7,9,14). SAM domains have also been described in interactions with non-SAM domain-containing proteins. For example, the SAM domain of BAR (46), a protein involved in the regulation of apoptosis, associates with both Bcl-2 and Bcl-XL (13). Cdk10, a member of the Cdc2 family of kinases, binds the SAM domain of Ets-2 and thereby regulates the activity of this transcription factor (15). The mitogen-activated protein kinase Erk2 docks on the SAM domain of Ets-1, enhancing the kinetics of phosphorylation at an adjacent N-terminal target site within this transcription factor (16). Although several complexes between nonidentical SAM domains have been described like TEL/TEL2 (17-23), and Yan/Mae (24) Scm/ph (3,11,25), their recognition mechanisms have not yet been characterized. Here we investigate one example of a hetero-SAM domain interaction that occurs between the Byr2 and Ste4 proteins in the fission yeast Schizosaccharomyces pombe.Sexual differentiation in S. pombe is controlled via a mitogen-activated protein kinase pathway that includes Ste4 and Byr2 (26). Byr2 is a mitogen-activated protein kinase kinase kinase that is activated by interactions with both . The SAM domain of Byr2 has previously been shown to bind to the N-terminal 160 amino acids of Ste4, a region containing a SAM domain followed immediately by a putative leucine zipper (Ste4-LZ) domain. A speculative model of Byr2 activation has therefore emerged in which Byr2 and Ste4 interact via their SAM domains, leading to oligomerization of Byr2 by virtue of the Ste4 leucine zipper domain (30). Here we find that although the two SAM domains do bind to each other, the role of the Ste4-LZ domain is not to oligomerize Byr2. Instead, the leucine zipper domain of Ste4 trimerizes, thereby displaying three SAM domains that together bind a single Byr2-SAM domain with high affinity.
EXPERIMENTAL PROCEDURESSte4 and Byr2 Constructs-The region of the Byr2 gene encompassing its SAM domain (amino acids 1-70; SPBC1D7.05 in the S. pombe GeneDB, www.genedb.org/genedb/pombe/index.jsp) was PCR-amplified from a S. pombe cDNA library and cloned into a modified pET-3c (Novagen) expression vector containing a C-terminal six-histidine tag. The expressed protein sequence comprised amino acids 1-70 of Byr2, followed by RDHHHHHH. The DNA sequences encoding the SAM domain (amino acids 9 -72) and the...
