Control of meristem development by CLAVATA1 receptor kinase and kinase-associated protein phosphatase interactions

Abstract

The CLAVATA1 (CLV1) gene encodes a putative receptor kinase required for the proper balance between cell proliferation and differentiation in Arabidopsis shoot and flower meristems. Impaired CLV1 signaling results in masses of undifferentiated cells at the shoot and floral meristems. Although many putative receptor kinases have been identified in plants, the mechanism of signal transduction mediated by plant receptor-like kinases is largely unknown. One potential effector of receptor kinase signaling is kinase-associated protein phosphatase (KAPP), a protein that binds to multiple plant receptor-like kinases in a phosphorylation-dependent manner. To examine a possible role for KAPP in CLV1-dependent plant development, the interaction of CLV1 and KAPP was investigated in vitro and in vivo. KAPP binds directly to autophosphorylated CLV1 in vitro and co-immunoprecipitates with CLV1 in plant extracts derived from meristematic tissue. Reduction of KAPP transcript accumulation in an intermediate clv1 mutant suppresses the mutant phenotype, and the degree of suppression is inversely correlated with KAPP mRNA levels. These data suggest that KAPP functions as a negative regulator of CLV1 signaling in plant development. This may represent a general model for the interaction of KAPP with receptor kinases.

Figure 1

CLV1 encoded an active protein kinase that autophosphorylates on multiple Ser residues. A, MBP fusions to CLV1CAT and CLV1CAT(K720E), which contains a point mutation at the conserved Lys required for phosphotransfer, were expressed in E. coli. Recombinant MBP-CLV1CAT is capable of incorporation of ³²P when incubated with [γ-³²P]ATP, indicating that it autophosphorylates (lanes 2), whereas the mutated version, MBP-CLV1CAT(K720E), is inactive (lanes 1). On the left is a Coomassie blue-stained SDS-PAGE gel and on the right the corresponding autoradiogram. Sizes of molecular mass standards in kilodaltons are indicated on the left. B, Autophosphorylation of MBP-CLV1CAT occurred on Ser residues. Autophosphorylated MBP-CLV1CAT was excised from an SDS-PAGE gel, hydrolyzed to individual amino acids in 6 n HCl, separated by two-dimensional TLE, and exposed to film. Positions of PAA standards (PSer, PThr, and PTyr) are indicated. C, Autophosphorylation of MBP-CLV1CAT occurred at multiple sites. Autophosphorylated MBP-CLV1CAT was excised from an SDS-PAGE gel, treated with trypsin, separated by two-dimensional TLE/TLC, and exposed to film. Tryptic peptides were applied to a cellulose plate (+) and resolved by electrophoresis in pH-1.9 buffer, followed by ascending chromatography. {/ANNT;;;left;top}

Figure 2

The KI domain of KAPP interacted with the autophosphorylated form of CLV1. Affinity-purified recombinant fusion proteins were subjected to SDS-PAGE, electrophoretically transferred to PVDF, and probed with ³²P-labeled fusion protein containing the KI domain. Molecular mass markers are indicated on the left in kilodaltons. On the left is a gel stained with Coomassie blue and on the right the corresponding autoradiogram after probing with GST-KI. The KI domain was capable of interacting with the catalytic domain of CLV1 (MBP-CLV1CAT) (lanes 2); however, the KI domain could not bind to the mutated form of CLV1 (MBP-CLV1CAT[K720E]) (lanes 1), which was incapable of autophosphorylation. Control blots probed with ³²P-labeled GST showed no binding.

Figure 3

KAPP and CLV1 associated in vivo. Antibodies raised to recombinant KAPP and CLV1 were used to co-immunoprecipitate the proteins from cauliflower meristem extracts. A and B, Demonstration of the specificity of CLV1 antibodies. Extracts (25 μg lane⁻¹) from mutant (clv1-6) and wild-type (Landsberg erecta [Ler]) Arabidopsis inflorescence tissue, including mature flowers, cauline leaves, and some stem tissue, were resolved by SDS-PAGE. A, Coomassie blue-stained gel. B, Results of chemiluminescence detection of immunoreactive polypeptides after incubation with CLV1 antisera. C, Proteins extracted from cauliflower (Caul.) and Arabidopsis (Arab.) were subjected to immunodetection using affinity-purified KAPP antibodies. D and E, Cauliflower meristematic tissue extracts were incubated with immobilized preimmune (PI) or immune (I) antisera against CLV1. Immunocomplexes were eluted, resolved by SDS-PAGE, and immunodetected with immune antisera against CLV1 (D) or KAPP (E). The positions of molecular mass standards are indicated on the left of each panel in kilodaltons.

Figure 4

Suppression of the clv1-1 phenotype correlated with a reduction in KAPP mRNA levels. A, Comparison of typical siliques from a wild-type plant (Landsberg erecta [Ler]) and three clv1-1 plants transformed with a 35S KAPP construct exhibiting varying degrees of suppression of the clv1 phenotype: complete, partial, and none. B, Analysis of RNA isolated from the corresponding transgenic plants shown in A. RNA was isolated from inflorescence tissue, separated in a formaldehyde-containing agarose gel, blotted to Hybond-N membranes, and hybridized with a ³²P-labeled KAPP cDNA probe. The membrane was also probed with 18S rRNA as a loading control. On the left is an autoradiogram of the RNA blots and on the right a graph showing the relative levels of KAPP transcript determined by quantitation of the blots. KAPP mRNA levels are expressed as a percentage of that in plants exhibiting no suppression. C, Partially (Partial) and completely (Complete) suppressed 35S KAPP plants accumulated reduced levels of KAPP mRNA compared with untransformed wild-type plants (WT). D, KAPP mRNA levels were equivalent in wild-type (WT) and clv1-1 inflorescence tissues. On the left is an autoradiogram of RNA blots hybridized with KAPP cDNA and 18S rRNA as a loading control and on the right a graph showing the relative levels of KAPP mRNA determined by quantitation of the blots. RNA analysis was repeated with similar results.