The integral membrane S-locus receptor kinase of Brassica has serine/threonine kinase activity in a membranous environment and spontaneously forms oligomers in planta

Abstract

To gain further insight into the mode of action of S-locus receptor kinase (SRK), a receptor-like kinase involved in the self-incompatibility response in Brassica, different recombinant SRK proteins have been expressed in a membranous environment using the insect cell͞baculovirus system. Recombinant SRK proteins exhibited properties close to those of the endogenous stigmatic SRK protein and were found to autophosphorylate on serine and threonine residues in insect cell microsomes. Autophosphorylation was constitutive because it did not require the presence of pollen or stigma extracts in the phosphorylation buffer. Phosphorylation was shown to occur in trans, suggesting the existence of constitutive homooligomers of membrane-anchored recombinant SRK. To investigate the physiological relevance of these results, we have examined the oligomeric status of SRK in planta in cross-linking experiments and by velocity sedimentation on sucrose gradients. Our data strongly suggest that SRK is associated both with other SRK molecules and other stigma proteins in nonpollinated flowers. These findings may have important implications for our understanding of self-pollen signaling. baculovirus ͉ oligomerization ͉ sporophytic self-incompatibility I n the Brassica family, the cell-cell interaction that leads to the rejection of self-pollen at the stigmatic surface [the selfincompatibility (SI) reaction] is controlled genetically by the multiallelic S-locus (1). When the pollen parent and the stigma share common S haplotypes, pollen germination or pollen tube growth is inhibited, thereby preventing self-fertilization. Several genes have been localized at the S locus but only two, the S-locus glycoprotein (SLG) and the S-locus receptor kinase (SRK) genes, exhibit the polymorphic nature expected for genes involved in the SI response (2, 3). SLG encodes a secreted glycoprotein that accumulates in the cell wall of papillae (4), whereas SRK encodes a plasmalemma-anchored glycoprotein (5, 6). SRK is structurally analogous to animal receptor kinases and belongs to the plant receptor-like kinase (RLK) family (7). Although its membrane topology has not been experimentally defined, DNA sequence analysis predicts that SRK consists of three domains: an extracellular glycosylated N-terminal domain (the S-domain) that shares extensive homology with SLG, a membrane-spanning domain, and a cytoplasmic domain (3). The cytoplasmic domain has been shown to have serine͞threonine kinase activity when expressed in bacteria (8). In the S 3 haplotype, SRK has been shown to encode, in addition to the integral membrane SRK protein, a soluble truncated form corresponding to the S-domain of SRK, the eSRK protein (9).Although there is some controversy as to whether SLG is required for the SI response (10, 11), there is strong evidence that SRK is necessary. For example, SRK was shown to be mutated in two Brassica lines that exhibited a self-compatible phenotype (12, 13). Moreover, Stahl and coworkers (14) recently have shown that the SI phenotype was altered i...

Mechanisms of self-incompatibility in flowering plants

“…SRK has been found to be membrane associated, as predicted for a receptor kinase, and has a functional serine/threonine kinase domain [112, 116 -119]. Giranton et al [119] conducted further biochemical studies on SRK using an insect/baculovirus system, and found that the phosphorylation of the recombinant SRK integral membrane protein was constitutive in the absence of pollen or stigma extracts. SRK phosphorylation was shown to occur in trans, whereby a kinaseinactive SRK became phosphorylated when co-expressed with a functional SRK construct likely as a result of SRK dimerization [119].…”

“…Giranton et al [119] conducted further biochemical studies on SRK using an insect/baculovirus system, and found that the phosphorylation of the recombinant SRK integral membrane protein was constitutive in the absence of pollen or stigma extracts. SRK phosphorylation was shown to occur in trans, whereby a kinaseinactive SRK became phosphorylated when co-expressed with a functional SRK construct likely as a result of SRK dimerization [119]. Experiments performed to address the physiological relevance of these results examined the oligomeric status of SRK in planta and showed that SRK forms complexes with either other SRK/SLGs or other stigmatic proteins in unpollinated flowers [119].…”

“…SRK phosphorylation was shown to occur in trans, whereby a kinaseinactive SRK became phosphorylated when co-expressed with a functional SRK construct likely as a result of SRK dimerization [119]. Experiments performed to address the physiological relevance of these results examined the oligomeric status of SRK in planta and showed that SRK forms complexes with either other SRK/SLGs or other stigmatic proteins in unpollinated flowers [119].…”

“…(B) Model for the rejection of self-incompatible pollen in Brassica. In an incompatible pollination, the SP11/SCR ligand is proposed to bind to and activate SRK, suggested to exist as oligomeric complexes in the stigmatic membranes [119]. In the absence of incompatible pollen, SRK is proposed to be inhibited by THL1 (and THL2), and this inhibition is released upon the addition of haplotypespecific SP11/SCR pollen ligand.…”

“…Vol. 58,2001 Review Article constitutively active [119]. When a soluble stigmatic extract was added to membrane fractions purified from either stigmas or from the SRK expressing insect/baculovirus system, SRK no longer had constitutive phosphorylation activity.…”