Vaka S. Reddy

Genes encoding calmodulin-binding proteins in the Arabidopsis genome

Analysis of the recently completedArabidopsis genome sequence indicates that ∼31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca2+ sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms fromArabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca2+-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching theArabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2–20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest thatArabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca2+-mediated signaling networks.

Analysis of EF-hand-containing proteins in Arabidopsis

BackgroundIn plants, calcium (Ca2+) has emerged as an important messenger mediating the action of many hormonal and environmental signals, including biotic and abiotic stresses. Many different signals raise cytosolic calcium concentration ([Ca2+]cyt), which in turn is thought to regulate cellular and developmental processes via Ca2+-binding proteins. Three out of the four classes of Ca2+-binding proteins in plants contain Ca2+-binding EF-hand motif(s). This motif is a conserved helix-loop-helix structure that can bind a single Ca2+ ion. To identify all EF-hand-containing proteins in Arabidopsis, we analyzed its completed genome sequence for genes encoding EF-hand-containing proteins.ResultsA maximum of 250 proteins possibly having EF-hands were identified. Diverse proteins, including enzymes, proteins involved in transcription and translation, protein- and nucleic-acid-binding proteins and a large number of unknown proteins, have one or more putative EF-hands. Phylogenetic analysis identified six major groups that contain some families of proteins.ConclusionsThe presence of EF-hand motif(s) in a diversity of proteins is consistent with the involvement of Ca2+ in regulating many cellular and developmental processes. Thus far, only 47 of the possible 250 EF-hand proteins have been reported in the literature. Various domains that we identified in many of the uncharacterized EF-hand-containing proteins should help in elucidating their cellular role(s). Our analyses suggest that the Ca2+ messenger system is widely used in plants and that EF-hand-containing proteins are likely to be the key transducers mediating Ca2+ action.

KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis

Kinesin-like calmodulin binding protein (KCBP) is a microtubule motor protein involved in the regulation of cell division and trichome morphogenesis. Genetic studies have shown that KCBP is likely to interact with several other proteins. To identify KCBP-interacting proteins, we used the C-terminal region of KCBP in a yeast two-hybrid screen. This screening resulted in the isolation of a novel KCBP-interacting Ca2+ binding protein (KIC). KIC, with its single EF-hand motif, bound Ca2+ at a physiological concentration. Coprecipitation with bacterially expressed protein and native KCBP, gel-mobility shift studies, and ATPase assays with the KCBP motor confirmed that KIC interacts with KCBP in a Ca2+-dependent manner. Interestingly, although both Ca2+-KIC and Ca2+-calmodulin were able to interact with KCBP and inhibit its microtubule binding activity, the concentration of Ca2+ required to inhibit the microtubule-stimulated ATPase activity of KCBP by KIC was threefold less than that required for calmodulin. Two KIC-related Ca2+ binding proteins and a centrin from Arabidopsis, which contain one and four EF-hand motifs, respectively, bound Ca2+ but did not affect microtubule binding and microtubule-stimulated ATPase activities of KCBP, indicating the specificity of Ca2+ sensors in regulating their targets. Overexpression of KIC in Arabidopsis resulted in trichomes with reduced branch number resembling the zwichel/kcbp phenotype. These results suggest that KIC modulates the activity of KCBP in response to changes in cytosolic Ca2+ and regulates trichome morphogenesis.

Interaction of a Kinesin-like Protein with Calmodulin Isoforms from Arabidopsis*

In Arabidopsis and other plants there are multiple calmodulin isoforms. However, the role of these isoforms in regulating the activity of target proteins is obscure. Here, we analyzed the interaction between a kinesin-like calmodulin-binding motor protein (Reddy, A. S. N., Safadi, F., Narasimhulu, S. B., Golovkin, M., and Hu, X. (1996) J. Biol. Chem. 271, 7052–7060) and three calmodulin isoforms (calmodulin-2, -4, and -6) from Arabidopsis using different approaches. Gel mobility and fluorescence shift assays revealed that the motor binds to all calmodulin isoforms in a calcium-dependent manner. Furthermore, all calmodulin isoforms were able to activate bovine calcium/calmodulin-dependent phosphodiesterase. However, the concentration of calmodulin-2 required for half-maximal activation of phosphodiesterase is 2- and 6-fold lower compared with calmodulin-4 and -6, respectively. The dissociation constants of the motor to calmodulin-2, -4, and -6 are 12.8, 27.0, and 27.8 nm, respectively, indicating that calmodulin-2 has 2-fold higher affinity for the motor than calmodulin-4 and -6. Similar results were obtained using another assay that involves the binding of35S-labeled calmodulin isoforms to the motor. The binding saturation curves of the motor with calmodulin isoforms have confirmed that calmodulin-2 has 2-fold higher affinity to the motor. However, the affinity of calmodulin-4 and -6 isoforms for the motor was about the same. Based on these studies, we conclude that all calmodulin isoforms bind to the motor protein but with different affinities.

Differential expression of genes encoding calmodulin-binding proteins in response to bacterial pathogens and inducers of defense responses

Calmodulin (CaM) plays an important role in sensing and transducing changes in cellular Ca2+ concentration in response to several biotic and abiotic stresses. Although CaM is implicated in plant-pathogen interactions, its molecular targets and their role in defense signaling pathway(s) are poorly understood. To elucidate the signaling pathways that link CaM to defense responses, we screened a cDNA library constructed from bean leaves undergoing a hypersensitive response (HR) with radiolabeled CaM isoforms. A total of 26 putative CBPs were identified. Sequencing of the cDNAs revealed that they represent 8 different genes. They are homologues of previously identified CaM-binding proteins (CBPs) in other systems. However, some CBPs are novel members of known CBP families. The proteins encoded by these clones bound CaM in a Ca2+-dependent manner. To determine if these CBPs are involved in plant defense responses, we analyzed their expression in bean leaves inoculated with compatible, incompatible and nonpathogenic bacterial strains. Expression of three CBPs including an isoform of cyclic nucleotide-gated channels (PvCNGC-A) and two hypothetical proteins (PvCBP60-C and PvCBP60-D) was induced whereas the expression of two other isoforms of CNGCs (PvCNGC-Band PvCNGC-C) was repressed in response to incompatible pathogens. The expression of the rest, a small auxin up RNA (PvSAUR1) and two hypothetical proteins (PvCBP60-Aand PvCBP60-B), was not changed. The expression of most of the pathogen-regulated genes was also affected by salicylic acid, jasmonic acid, hydrogen peroxide and a fungal elicitor, which are known to induce defense responses. Our results strongly suggest that at least five bean CBPs are involved in plant defense responses.

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca2+ at the pollen tube tip and its growth. Since the action of Ca2+ is primarily mediated by Ca2+-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca2+regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maizepollen calmodulin-bindingprotein, MPCBP) was isolated in a protein-protein interaction-based screening using 35S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins fromArabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca2+-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca2+-regulated events during pollen germination and growth.

A plant calmodulin-binding motor is part kinesin and part myosin.

Kinesins and myosins are molecular motors that move on microtubules and actin filaments, respectively. These motor proteins are involved in a variety of cellular processes such as intracellular transport, spindle formation and chromosome segregation in eukaryotes (Langford, 1995; Hirokawa, 1998). Although kinesins and myosins hydrolyze ATP to generate force for their movement on cytoskeletal filaments and have similar structural organization (head/motor, stalk and tail) there is virtually no amino acid (aa) sequence identity between the microtubuleand actin-based motors (Kull et al., 1996). We report here that a novel calmodulin-binding protein from plants has domains that are present in microtubuleand actin-based motors, suggesting that this class of plant motors is unique in having kinesin and myosin features. In a protein–protein interaction based screening with calmodulin we recently isolated a cDNA from Arabidopsis encoding a novel calmodulin (CaM)-binding protein, KCBP, with a kinesin-like motor domain at the C-terminus of the protein (Reddy et al., 1996). Motility studies with the C-terminal kinesin-like motor domain alone have shown that it is a minus-end directed microtubule motor (Song et al., 1997). KCBP is unique among all known kinesins and kinesin-like proteins in having a CaMbinding domain (CBD). The homolog of KCBP has been cloned from three other plant systems, suggesting that it is ubiquitous in flowering plants (Narasimhulu and Reddy, 1998). However, a KCBP homolog has not been found in yeast and Caenorhabditis elegans, whose genomes have been completely sequenced, or in any other non-plant systems. In our ongoing analysis of KCBP function, we analyzed KCBP sequence using BLASTP and SMART programs (http://coot.embl-heidelberg.de/SMART) and identified, in addition to kinesin-like motor domain at the C-terminus, a region that is conserved in the tails of some members of myosins (Figure 1). The C-terminal region (amino acids 860–1217) of KCBP showed similarities with the motor region of a large number of microtubulebased motors of which KIFC3, a C-terminal motor, showed the highest sequence similarity (43% identity and 56% similarity, Figure 1A). The tail of myosin VIIa has two long repeats (about ∼460 aa per repeat), each containing a MyTH4 (myosin tail homology 4) domain (∼110 aa) and a talin-like (∼350 aa) domain. Although the MyTH4 domain and the talin-like regions exist singly in some myosins (IV and XII) and in the band 4.1 superfamily (examples include talin and ERM proteins), respectively, the presence of these two regions together (MyTH4+talin-like) occurs only in myosin VIIa (as two repeats) and myosin X (one repeat) (Chen et al., 1996). The N-terminal tail (aa 121–612) of KCBP showed significant similarities to the MyTH4 and talin-like (band 4.1) regions present in myosins VIIa and X. The MyTH4 and the talin-like domains of KCBP showed the highest similarity with the myosin VIIa tail (29% identity and 45% similarity in the MyTH4 domain and 23% identity and 37% similarity in the talin-like region) (Figure 1B). The MyTH4 domain and talin-like region have not been found in any other known members of the kinesin superfamily. Therefore, we conclude that the KCBP is a molecular hybrid consisting of a motor domain from microtubule-based motors and a tail region of actin-based motors (Figure 1C). The significance of MyTH4 and talin-like domains in KCBP and myosins is not known at this time, but the existence of talin-like region together with MyTH4 domain in KCBP, myosin VIIa and X is interesting and points to some functional significance. Based on what is known about KCBP and myosin VIIa, we propose that these domains may be involved in one or more of the following functions. One possibility is that the KCBP and myosin VIIa-like proteins could interact with some unknown common protein(s) through their tail homology regions to either cross-bridge microtubule and actin filaments or facilitate cargo exchange between these two types of molecular motors. An elegant study by Lille and Brown (1992) showed that a lethal mutation resulting from a defect in a myosin motor could be complemented by a kinesin motor protein which also suggests that actinand microtubule-motors are likely to interact with some common set of proteins. Furthermore,

Characterization of a pathogen-induced calmodulin-binding protein: mapping of four Ca2+-dependent calmodulin-binding domains.

Ca2+ and calmodulin (CaM), a key Ca2+ sensor in all eukaryotes, have been implicated in defense responses in plants. To elucidate the role of Ca2+ and CaM in defense signaling, we used 35S-labeled CaM to screen expression libraries prepared from tissues that were either treated with an elicitor derived from Phytophthora megasperma or infected with Pseudomonas syringae pv. tabaci. Nineteen cDNAs that encode the same protein, pathogen-induced CaM-binding protein (PICBP), were isolated. The PICBP fusion proteins bound 35S-CaM, horseradish peroxidase-labeled CaM and CaM-Sepharose in the presence of Ca2+ whereas EGTA, a Ca2+ chelator, abolished binding, confirming that PICBP binds CaM in a Ca2+-dependent manner. Using a series of bacterially expressed truncated versions of PICBP, four CaM-binding domains, with a potential CaM-binding consensus sequence of WSNLKKVILLKRFVKSL, were identified. The deduced PICBP protein sequence is rich in leucine residues and contains three classes of repeats. The PICBP gene is differentially expressed in tissues with the highest expression in stem. The expression of PICBP in Arabidopsis was induced in response to avirulent Pseudomonas syringae pv. tomato carrying avrRpm1. Furthermore, PICBP is constitutively expressed in the Arabidopsisaccelerated cell death2-2 mutant. The expression of PICBP in bean leaves was also induced after inoculation with avirulent and non-pathogenic bacterial strains. In addition, the hrp1 mutant of Pseudomonas syringae pv. tabaci and inducers of plant defense such as salicylic acid, hydrogen peroxide and a fungal elicitor induced PICBP expression in bean. Our data suggest a role for PICBP in Ca2+-mediated defense signaling and cell-death. Furthermore, PICBP is the first identified CBP in eukaryotes with four Ca2+-dependent CaM-binding domains.

The Calmodulin-binding Domain from a Plant Kinesin Functions as a Modular Domain in Conferring Ca2+-Calmodulin Regulation to Animal Plus- and Minus-end Kinesins

Plant kinesin-like calmodulin-binding protein (KCBP) is a novel member of the kinesin superfamily that interacts with calmodulin (CaM) via its CaM-binding domain (CBD). Activated CaM (Ca2+-CaM) has been shown to inhibit KCBP interaction with microtubules (MTs) thereby abolishing its motor- and MT-dependent ATPase activities. To test whether the fusion of CBD to non-CaM-binding kinesins confers Ca2+-CaM regulation, we fused the CBD of KCBP to the N or C terminus of a minus-end (non-claret disjunction) or C terminus of a plus-end (Drosophila kinesin) motor. Purified chimeric kinesins bound CaM in a Ca2+-dependent manner whereas non-claret disjunction, Drosophila kinesin, and KCBP that lack a CBD did not. As in the case of KCBP with CBD, the interaction of chimeric motors with MTs, as well as their MT-stimulated ATPase activity, was inhibited by Ca2+-CaM. The presence of a spacer between the motor and CBD did not alter Ca2+-CaM regulation. However, KCBP interaction with MTs and its MT-stimulated ATPase activity were not inhibited when the motor domain and CBD were added separately, suggesting that Ca2+-CaM regulation of CaM-binding motors occurs only when the CBD is attached to the motor domain. These results show that the fusion of the CBD to animal motors confers Ca2+-CaM regulation and suggest that the CBD functions as a modular domain in disrupting motor-MT interaction. Our data also support the hypothesis that CaM-binding kinesins may have evolved by addition of a CBD to a kinesin motor domain.

Structural dynamics of the microtubule binding and regulatory elements in the kinesin-like calmodulin binding protein.

Kinesins are molecular motors that power cell division and transport of various proteins and organelles. Their motor activity is driven by ATP hydrolysis and depends on interactions with microtubule tracks. Essential steps in kinesin movement rely on controlled alternate binding to and detaching from the microtubules. The conformational changes in the kinesin motors induced by nucleotide and microtubule binding are coordinated by structural elements within their motor domains. Loop L11 of the kinesin motor domain interacts with the microtubule and is implicated in both microtubule binding and sensing nucleotide bound to the active site of kinesin. Consistent with its proposed role as a microtubule sensor, loop L11 is rarely seen in crystal structures of unattached kinesins. Here, we report four structures of a regulated plant kinesin, the kinesin-like calmodulin binding protein (KCBP), determined by X-ray crystallography. Although all structures reveal the kinesin motor in the ATP-like conformation, its loop L11 is observed in different conformational states, both ordered and disordered. When structured, loop L11 adds three additional helical turns to the N-terminal part of the following helix alpha4. Although interactions with protein neighbors in the crystal support the ordering of loop L11, its observed conformation suggests the conformation for loop L11 in the microtubule-bound kinesin. Variations in the positions of other features of these kinesins were observed. A critical regulatory element of this kinesin, the calmodulin binding helix positioned at the C-terminus of the motor domain, is thought to confer negative regulation of KCBP. Calmodulin binds to this helix and inserts itself between the motor and the microtubule. Comparison of five independent structures of KCBP shows that the positioning of the calmodulin binding helix is not decided by crystal packing forces but is determined by the conformational state of the motor. The observed variations in the position of the calmodulin binding helix fit the regulatory mechanism previously proposed for this kinesin motor.