Biochemical characterization of domains in the membrane subunit DrrB that interact with the ABC subunit DrrA: identification of a conserved motif

DrrA and DrrB proteins confer resistance to the commonly used anticancer agents daunorubicin and doxorubicin in the producer organism Streptomyces peucetius. The drrAB locus has previously been cloned in Escherichia coli, and the proteins have been found to be functional in this host. DrrA, a soluble protein, belongs to the ABC family of proteins. It forms a complex with the integral membrane protein DrrB. Previous studies suggest that the function and stability of DrrA and DrrB are biochemically coupled. Thus, DrrA binds ATP only when it is in a complex with DrrB in the membrane. Further, DrrB is completely degraded if DrrA is absent. In the present study, we have characterized domains in DrrB that may be directly involved in interaction with DrrA. Several single-cysteine substitutions in DrrB were made. Interaction between DrrA and DrrB was studied by using a cysteine to amine chemical cross-linker that specifically cross-links a free sulfhydryl group in one protein (DrrB) to an amine in another (DrrA). We show here that DrrA cross-links with both the N- and the C-terminal ends of the DrrB protein, implying that they may be involved in interaction. Furthermore, this study identifies a motif within the N-terminal cytoplasmic tail of DrrB, which is similar to a motif recently shown by crystal structure analysis in BtuC and previously shown by sequence analysis to be also present in exporters, including MDR1. We propose that the motif present in DrrB and other exporters is actually a modified version of the EAA motif, which was originally believed to be present only in the importers of the ABC family. The present work is the first report where domains of interaction in the membrane component of an ABC drug exporter have been biochemically characterized.     

The Extreme C Terminus of the ABC Protein DrrA Contains Unique Motifs Involved in Function and Assembly of the DrrAB Complex

Two novel regulatory motifs, LDEVFL and C-terminal regulatory Glu (E)-rich motif (CREEM), are identified in the extreme C terminus of the ABC protein DrrA, which is involved in direct interaction with the N-terminal cytoplasmic tail of the membrane protein DrrB and in homodimerization of DrrA. Disulfide cross-linking analysis showed that the CREEM and the region immediately upstream of CREEM participate directly in forming an interaction interface with the N terminus of DrrB. A series of mutations created in the LDEVFL and CREEM motifs drastically affected overall function of the DrrAB transporter. Mutations in the LDEVFL motif also significantly impaired interaction between the C terminus of DrrA and the N terminus of DrrB as well as the ability of DrrA and DrrB to co-purify, therefore suggesting that the LDEVFL motif regulates CREEM-mediated interaction between DrrA and DrrB and plays a key role in biogenesis of the DrrAB complex. Modeling analysis indicated that the LDEVFL motif is critical for conformational integrity of the C-terminal domain of DrrA and confirmed that the C terminus of DrrA forms an independent domain. This is the first report which describes the presence of an assembly domain in an ABC protein and uncovers a novel mechanism whereby the ABC component facilitates the assembly of the membrane component. Homology sequence comparisons showed the presence of the LDEVFL and CREEM motifs in close prokaryotic and eukaryotic homologs of DrrA, suggesting that these motifs may play a similar role in other homologous drug and lipid export systems.     

Expression and characterization of DrrA and DrrB proteins of Streptomyces peucetius in Escherichia coli: DrrA is an ATP binding protein.

Streptomyces peucetius, a microorganism that produces the anticancer drugs doxorubicin and daunorubicin, is itself resistant to the action of these drugs. The genes conferring resistance to doxorubicin and daunorubicin in S. peucetius have been sequenced (P. G. Guilfoile and R. Hutchinson, Proc. Natl. Acad. Sci. USA 88:8553-8557, 1991). Two open reading frames, drrA and drrB, were proposed to encode for an ABC (ATP-binding cassette) type of permease that carries out export of the antibiotics in an ATP-dependent manner. This article reports subcloning of the drrA and drrB genes into Escherichia coli expression vectors and characterization of their gene products. Upon induction from the lac promoter, a 36-kDa DrrA protein could be identified on Coomassie blue-stained gels. The DrrB protein was identified by use of a polyclonal antiserum generated against a synthetic peptide corresponding to a portion of the DrrB protein. Together, the DrrA and DrrB proteins conferred resistance to doxorubicin in E. coli. The DrrB protein was localized to the cell membrane. The DrrA protein bound ATP or GTP in a Mg2+-dependent fashion. ATP binding was enhanced on addition of doxorubicin or daunorubicin.     

Membrane topology of the DrrB protein of the doxorubicin transporter of Streptomyces peucetius

Daunorubicin and doxorubicin, two commonly used anticancer agents, are produced by the soil bacterium Streptomyces peucetius. Self-resistance to these antibiotics in S. peucetius is conferred by the drrAB locus that codes for two proteins, DrrA and DrrB. DrrA is an ATP-binding protein. It belongs to the ABC family of transporters and shares sequence and functional similarities with P-glycoprotein of cancer cells. DrrB is an integral membrane protein that might function as a transporter for the efflux of daunorubicin and doxorubicin. Together, DrrA and DrrB are believed to form an ATP-driven pump for the efflux of these drugs. The drrAB locus has been cloned, and the two proteins have been expressed in a functional form in Escherichia coli. A topological analysis of the DrrB protein was performed using gene fusion methodology. Random and site-directed fusions of the drrB gene to lacZ, phoA, or gfp reporter genes were created. Based on the fusion data, a topological model of the DrrB protein is proposed in which the protein has eight membrane-spanning domains with both the N terminus and the C terminus in the cytoplasm.     

Translational Coupling Controls Expression and Function of the DrrAB Drug Efflux Pump

This study investigates the role of translational coupling in the expression and function of DrrA and DrrB proteins, which form an efflux pump for the export of anticancer drugs doxorubicin and daunorubicin in the producer organism Streptomyces peucetius. Interest in studying the role of translational coupling came from the initial observation that DrrA and DrrB proteins confer doxorubicin resistance only when they are expressed in cis. Because of the presence of overlapping stop and start codons in the intergenic region between drrA and drrB, it has been assumed that the translation of drrB is coupled to the translation of the upstream gene drrA even though direct evidence for coupling has been lacking. In this study, we show that the expression of drrB is indeed coupled to translation of drrA. We also show that the introduction of non-coding sequences between the stop codon of drrA and the start of drrB prevents formation of a functional complex, although both proteins are still produced at normal levels, thus suggesting that translational coupling also plays a crucial role in proper assembly. Interestingly, replacement of drrA with an unrelated gene was found to result in very high drrB expression, which becomes severely growth inhibitory. This indicates that an additional mechanism within drrA may optimize expression of drrB. Based on the observations reported here, it is proposed that the production and assembly of DrrA and DrrB are tightly linked. Furthermore, we propose that the key to assembly of the DrrAB complex lies in co-folding of the two proteins, which requires that the genes be maintained in cis in a translationally coupled manner.     

Characterization of the genes and proteins of a two-component system from the hyperthermophilic bacterium Thermotoga maritima.

As a step towards studying representative members of the two-component family of signal transduction proteins, we have cloned genes encoding a histidine protein kinase and a response regulator from the hyperthermophilic bacterium Thermotoga maritima. The genes have been designated HpkA and drrA, respectively. The deduced HpkA sequence contains all five characteristic histidine protein kinase motifs with the same relative order and spacing found in the mesophilic bacterial proteins. A hydropathy profile indicates that HpkA possesses only one membrane-spanning segment located at the extreme N terminus. The N-terminal region of DrrA exhibits all of the characteristics of the conserved domains of mesophilic bacterial response regulators, and the C-terminal region shows high similarity to the OmpR-PhoB subfamily of DNA-binding proteins. Recombinant T. maritima proteins, truncated HpkA lacking the putative membrane-spanning N- terminal amino acids and DrrA, were expressed in Escherichia coli. Partial purification of T. maritima proteins was achieved by heat denaturation of E. coli host proteins. In an in vitro assay, truncated HpkA protein was autophosphorylated in the presence of ATP. Thus, the N-terminal hydrophobic region is not required for kinase activity. Phosphotransfer between truncated HpkA and DrrA was demonstrated in vitro with the partially purified proteins. The phosphorylation reactions were strongly temperature dependent. The results indicate that the recombinant T. maritima two-component proteins overexpressed in E. coli are stable as well as enzymatically active at elevated temperatures.     

Structure-function analysis of the gE-gI complex of feline herpesvirus: mapping of gI domains required for gE-gI interaction, intracellular transport, and cell-to-cell spread.

Alphaherpesvirus glycoproteins gE and gI form a noncovalently associated hetero-oligomeric complex, which is involved in cell-to-cell spread. In the absence of gI, feline herpesvirus (FHV) gE is transport incompetent and fully retained in the endoplasmic reticulum. Here, we assess the effect of progressive C-terminal truncations of FHV gI on the biosynthesis, intracellular transport, and function of the gE-gI complex. The truncated gI proteins were coexpressed with gE in the vaccinia virus-based vTF7-3 expression system. The results were corroborated and extended by studying FHV recombinants expressing truncated gI derivatives. The following conclusions can be drawn. (i) Deletion of the cytoplasmic tail, the transmembrane region plus the C-terminal half of the ectodomain of gI, does not affect intracellular transport of gE. Apparently, the N-terminal 166 residues of gI constitute a domain involved in gE-gI interaction. (ii) A region mediating stable association with gE is located within the N-terminal 93 residues of gI. (iii) The cytoplasmic domain of gI is not essential for gE-gI-mediated cell-to-cell transmission of FHV, as judged from plaque morphology. Deletion of the cytoplasmic tail of gI reduced plaque size by only 35%. (iv) Recombinants expressing the N-terminal 166 residues of gI display a small-plaque phenotype but produce larger plaques than recombinants with a disrupted gI gene. Thus, a complex consisting of gE and the N-terminal half of the gI ectodomain may retain residual biological activity. The implications of these findings for gE-gI interaction and function are discussed.     

The Legionella pneumophila effector DrrA is sufficient to stimulate SNARE-dependent membrane fusion

The intracellular bacterial pathogen Legionella pneumophila subverts host membrane transport pathways to promote fusion of vesicles exiting the endoplasmic reticulum (ER) with the pathogen-containing vacuole. During infection there is noncanonical pairing of the SNARE protein Sec22b on ER-derived vesicles with plasma membrane (PM)-localized syntaxin proteins on the vacuole. We show that the L.?pneumophila Rab1-targeting effector DrrA is sufficient to stimulate this noncanonical SNARE association and promote membrane fusion. DrrA activation of the Rab1 GTPase on PM-derived organelles stimulated the tethering of ER-derived vesicles with the PM-derived organelle, resulting in vesicle fusion through the pairing of Sec22b with the PM syntaxin proteins. Thus, the effector protein DrrA stimulates a host membrane transport pathway that enables ER-derived vesicles to remodel a PM-derived organelle, suggesting that Rab1 activation at the PM is sufficient to promote the recruitment and fusion of ER-derived vesicles.     

High‐affinity binding of phosphatidylinositol 4‐phosphate by Legionella pneumophila DrrA

The DrrA protein of Legionella pneumophila is involved in mistargeting of endoplasmic reticulum‐derived vesicles to Legionella‐containing vacuoles through recruitment of the small GTPase Rab1. To this effect, DrrA binds specifically to phosphatidylinositol 4‐phosphate (PtdIns(4)P) lipids on the cytosolic surface of the phagosomal membrane shortly after infection. In this study, we present the atomic structure of the PtdIns(4)P‐binding domain of a protein (DrrA) from a human pathogen. A detailed kinetic investigation of its interaction with PtdIns(4)P reveals that DrrA binds to this phospholipid with, as yet unprecedented, high affinity, suggesting that DrrA can sense a very low abundance of the lipid.     

Dissection of the TssB-TssC Interface during Type VI Secretion Sheath Complex Formation

The Type VI secretion system (T6SS) is a versatile machine that delivers toxins into either eukaryotic or bacterial cells. At a molecular level, the T6SS is composed of a membrane complex that anchors a long cytoplasmic tubular structure to the cell envelope. This structure is thought to resemble the tail of contractile bacteriophages. It is composed of the Hcp protein that assembles into hexameric rings stacked onto each other to form a tube similar to the phage tail tube. This tube is proposed to be wrapped by a structure called the sheath, composed of two proteins, TssB and TssC. It has been shown using fluorescence microscopy that the TssB and TssC proteins assemble into a tubular structure that cycles between long and short conformations suggesting that, similarly to the bacteriophage sheath, the T6SS sheath undergoes elongation and contraction events. The TssB and TssC proteins have been shown to interact and a specific α-helix of TssB is required for this interaction. Here, we confirm that the TssB and TssC proteins interact in enteroaggregative E. coli. We further show that this interaction requires the N-terminal region of TssC and the conserved α-helix of TssB. Using site-directed mutagenesis coupled to phenotypic analyses, we demonstrate that an hydrophobic motif located in the N-terminal region of this helix is required for interaction with TssC, sheath assembly and T6SS function.     

The cytoplasmic tail of L-selectin interacts with members of the Ezrin-Radixin-Moesin (ERM) family of proteins: cell activation-dependent binding of Moesin but not Ezrin.

L-selectin regulates the recruitment of naive lymphocytes from the bloodstream to secondary lymphoid organs, mediating their initial capture and subsequent rolling along high endothelial cell surface-expressed ligands in peripheral lymph nodes. In vivo, distribution of L-selectin and cell surface levels determine the tethering efficiency and rolling velocity of leukocytes, respectively. Treatment of naive lymphocytes with phorbol myristate acetate (PMA) induces rapid ectodomain proteolytic down-regulation (shedding) of surface L-selectin via a protein kinase C (PKC)-dependent pathway. In an attempt to isolate proteins that are involved in regulating L-selectin expression, an affinity column was constructed using the 17-amino acid cytoplasmic tail of L-selectin. Affinity purification of extracts from lymphocytes, pre-treated with or without PMA, allowed identification of proteins that interact with the affinity column under one condition but not the other. By using this approach, members of the Ezrin-Radixin-Moesin family of proteins were found to interact specifically with the cytoplasmic tail of L-selectin. Moesin from PMA-stimulated lymphocytes, but not from unstimulated lymphocytes, bound to L-selectin tail. In contrast, ezrin from unstimulated or PMA-stimulated lymphocytes associated with L-selectin tail with equal affinity. Furthermore, the PKC inhibitor Ro 31-8220 significantly reduced the interaction of moesin, but not ezrin, with L-selectin. Alanine mutations of membrane-proximal basic amino acid residues in the cytoplasmic domain of L-selectin identified arginine 357 as a critical residue for both ezrin and moesin interaction. Finally, BIAcore affinity analysis confirmed that N-terminal moesin interacts specifically with L-selectin cytoplasmic tail, with relatively high affinity (K(d) approximately 40 nm). Based on these findings, although moesin and ezrin bind to a similar region of the cytoplasmic tail of L-selectin, moesin binding is dependent on PKC activation, which suggests that ezrin and moesin are regulated differently in lymphocytes.     

The transmembrane domain of murine alpha-mannosidase IB is a major determinant of Golgi localization

Murine alpha1,2-mannosidase IB is a type II transmembrane protein localized to the Golgi apparatus where it is involved in the biogenesis of complex and hybrid N-glycans. This enzyme consists of a cytoplasmic tail, a transmembrane domain followed by a "stem" region and a large C-terminal catalytic domain. To analyze the determinants of targeting, we constructed various deletion mutants of murine alpha1,2-mannosidase IB as well as alpha1,2-mannosidase IB/yeast alpha1,2-mannosidase and alpha1,2-mannosidase IB/GFP chimeras and localized these proteins by fluorescence microscopy, when expressed transiently in COS7 cells. Replacing the catalytic domain of alpha1,2-mannosidase IB with that of the homologous yeast alpha1,2-mannosidase and deleting the "stem" region in this chimera had no effect on Golgi targeting, but caused increased cell surface localization. The N-terminal tagged protein lacking a catalytic domain was also localized to the Golgi. In the latter case, when the stem region was partially or completely removed, the protein was found in both the ER and the Golgi. A chimera consisting of the alpha1,2-mannosidase IB N-terminal region (cytoplasmic and transmembrane domains plus 10 amino acids of the "stem" region) and GFP was localized mainly to the Golgi. Deletion of 30 out of 35 amino acids in the cytoplasmic tail had no effect on Golgi localization. A GFP chimera lacking the entire cytoplasmic tail was found in both the ER and the Golgi. These results indicate that the transmembrane domain of alpha1,2-mannosidase IB is a major determinant of Golgi localization.     

Structural insights into the dual nucleotide exchange and GDI displacement activity of SidM/DrrA

GDP‐bound prenylated Rabs, sequestered by GDI (GDP dissociation inhibitor) in the cytosol, are delivered to destined sub‐cellular compartment and subsequently activated by GEFs (guanine nucleotide exchange factors) catalysing GDP‐to‐GTP exchange. The dissociation of GDI from Rabs is believed to require a GDF (GDI displacement factor). Only two RabGDFs, human PRA‐1 and Legionella pneumophila SidM/DrrA, have been identified so far and the molecular mechanism of GDF is elusive. Here, we present the structure of a SidM/DrrA fragment possessing dual GEF and GDF activity in complex with Rab1. SidM/DrrA reconfigures the Switch regions of the GTPase domain of Rab1, as eukaryotic GEFs do toward cognate Rabs. Structure‐based mutational analyses show that the surface of SidM/DrrA, catalysing nucleotide exchange, is involved in GDI1 displacement from prenylated Rab1:GDP. In comparison with an eukaryotic GEF TRAPP I, this bacterial GEF/GDF exhibits high binding affinity for Rab1 with GDP retained at the active site, which appears as the key feature for the GDF activity of the protein.     

Vaccinia virus A36R membrane protein provides a direct link between intracellular enveloped virions and the microtubule motor kinesin

Previous work demonstrated that intracellular enveloped vaccinia virus virions use microtubules to move from the site of membrane wrapping to the cell periphery. The mechanism and direction of intracellular virion movement predicted that viral proteins directly or indirectly interact with the microtubule motor protein kinesin. The yeast two-hybrid assay was used to test for interactions between the light chain of kinesin and the cytoplasmic tails from five viral envelope proteins. We found that the N-terminal tetratricopeptide repeat region of the kinesin light chain (KLC-TPR) interacted with the cytoplasmic tail of the viral A36R protein. A series of C- and N-terminal truncations of A36R further defined a region from residues 81 to 111 that was sufficient for interaction with KLC-TPR. Interactions were confirmed by using pull-down assays with purified glutathione S-transferase (GST)-A36R and (35)S-labeled KLC-TPR. The defined region on A36R for interaction with kinesin overlaps the recently defined region (residues 91 to 111) for interaction with the A33R envelope protein. The yeast three-hybrid system was used to demonstrate that expression of A33R interrupted the interaction between A36R and KLC-TPR, indicating that the binding of A36R is mutually exclusive to either A33R or kinesin. Pull-down assays with purified GST-A36R and (35)S-labeled KLC-TPR in the presence of competing A33R corroborated these findings. Collectively, these results demonstrated that the viral A36R protein interacts directly with the microtubule motor protein kinesin and that the viral protein A33R may regulate this interaction.     

The Legionella pneumophila effector protein DrrA is a Rab1 guanine nucleotide-exchange factor

The intracellular pathogen Legionella pneumophila avoids fusion with lysosomes and subverts membrane transport from the endoplasmic reticulum to create an organelle that supports bacterial replication. Transport of endoplasmic reticulum-derived vesicles to the Legionella-containing vacuole (LCV) requires bacterial proteins that are translocated into host cells by a type IV secretion apparatus called Dot/Icm. Recent observations have revealed recruitment of the host GTPase Rab1 to the LCV by a process requiring the Dot/Icm system. Here, a visual screen was used to identify L. pneumophila mutants with defects in Rab1 recruitment. One of the factors identified in this screen was DrrA, a new Dot/Icm substrate protein translocated into host cells. We show that DrrA is a potent and highly specific Rab1 guanine nucleotide-exchange factor (GEF). DrrA can disrupt Rab1-mediated secretory transport to the Golgi apparatus by competing with endogenous exchange factors to recruit and activate Rab1 on plasma membrane-derived organelles. These data establish that intracellular pathogens have the capacity to directly modulate the activation state of a specific member of the Rab family of GTPases and thus further our understanding of the mechanisms used by bacterial pathogens to manipulate host vesicular transport.     

Nicastrin interacts with gamma-secretase complex components via the N-terminal part of its transmembrane domain

Two secretases are involved in the generation of amyloid beta-peptide, the principal component of amyloid plaques in the brains of Alzheimer's disease patients. While beta-secretase is a classical aspartyl protease, gamma-secretase activity is associated with a high molecular weight complex. One of the complex components, which is critically required for gamma-secretase activity is nicastrin (NCT). Here we investigate the assembly of NCT into the gamma-secretase complex. NCT mutants either lacking the entire cytoplasmic tail, the cytoplasmic tail, and the transmembrane domain (TMD), or containing a set of heterologous TMDs were expressed in cells with strongly reduced levels of endogenous NCT. Maturation of exogenous NCT, gamma-secretase complex formation and proteolytic function was then investigated. This revealed that the cytoplasmic tail of NCT is dispensable for gamma-secretase complex assembly and function. In contrast, the authentic TMD of NCT is critically required for the interaction with gamma-secretase complex components and for formation of an active gamma-secretase complex. Neither soluble NCT lacking any membrane anchor nor NCT containing a heterologous TMD were inserted into the gamma-secretase complex. We identified the N-terminal region of the NCT TMD as a functionally important entity of NCT. These data thus demonstrate that NCT interacts with other gamma-secretase complex components via its TMD.     

Protein interaction profiling of the p97 adaptor UBXD1 points to a role for the complex in modulating ERGIC-53 trafficking

UBXD1 is a member of the poorly understood subfamily of p97 adaptors that do not harbor a ubiquitin association domain or bind ubiquitin-modified proteins. Of clinical importance, p97 mutants found in familial neurodegenerative conditions Inclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal Dementia and Amyotrophic Lateral Sclerosis are defective at interacting with UBXD1, indicating that functions regulated by a p97-UBXD1 complex are altered in these diseases. We have performed liquid chromatography-mass spectrometric analysis of UBXD1-interacting proteins to identify pathways in which UBXD1 functions. UBXD1 displays prominent association with ERGIC-53, a hexameric type I integral membrane protein that functions in protein trafficking. The UBXD1-ERGIC-53 interaction requires the N-terminal 10 residues of UBXD1 and the C-terminal cytoplasmic 12 amino acid tail of ERGIC-53. Use of p97 and E1 enzyme inhibitors indicate that complex formation between UBXD1 and ERGIC-53 requires the ATPase activity of p97, but not ubiquitin modification. We also performed SILAC-based quantitative proteomic profiling to identify ERGIC-53 interacting proteins. This analysis identified known (e.g. COPI subunits) and novel (Rab3GAP1/2 complex involved in the fusion of vesicles at the cell membrane) interactions that are also mediated through the C terminus of the protein. Immunoprecipitation and Western blotting analysis confirmed the proteomic interaction data and it also revealed that an UBXD1-Rab3GAP association requires the ERGIC-53 binding domain of UBXD1. Localization studies indicate that UBXD1 modules the sub-cellular trafficking of ERGIC-53, including promoting movement to the cell membrane. We propose that p97-UBXD1 modulates the trafficking of ERGIC-53-containing vesicles by controlling the interaction of transport factors with the cytoplasmic tail of ERGIC-53.     

Identification of a myosin VII-talin complex

Myosin VII (M7) plays a role in adhesion in both Dictyostelium and mammalian cells where it is a component of a complex of proteins that serve to link membrane receptors to the underlying actin cytoskeleton. The nature of this complex is not fully known, prompting a search for M7-binding proteins. Co-immunoprecipitation experiments reveal that Dictyostelium M7 (DdM7) interacts with talinA, an actin-binding protein with a known role in cell-substrate adhesion. No additional proteins are observed in the immunoprecipitate, indicating that the interaction is direct. The N-terminal region of the DdM7 tail that lies between the region of predicted coil and the first MyTH4 domain is found to harbor the talinA binding site. Localization experiments reveal that talinA does not serve as a membrane receptor for DdM7 and vice versa. These findings reveal that talinA is a major DdM7 binding partner and suggest that their interaction induces a conformational change in each that, in combination with membrane receptor binding, promotes the assembly of a high avidity receptor complex essential for adhesion of the cell to substrata.     

Interaction of a kinesin-like calmodulin-binding protein with a protein kinase

Kinesin-like calmodulin-binding protein (KCBP) is a novel member of the kinesin superfamily that is involved in cell division and trichome morphogenesis. KCBP is unique among all known kinesins in having a myosin tail homology-4 region in the N-terminal tail and a calmodulin-binding region following the motor domain. Calcium, through calmodulin, has been shown to negatively regulate the interaction of KCBP with microtubules. Here we have used the yeast two-hybrid system to identify the proteins that interact with the tail region of KCBP. A protein kinase (KCBP-interacting protein kinase (KIPK)) was found to interact specifically with the tail region of KCBP. KIPK is related to a group of protein kinases specific to plants that has an additional sequence between subdomains VII and VIII of the conserved C-terminal catalytic domain and an extensive N-terminal region. The catalytic domain alone of KIPK interacted weakly with the N-terminal KCBP protein but strongly with full-length KCBP, whereas the noncatalytic region did not interact with either protein. The interaction of KCBP with KIPK was confirmed using coprecipitation assays. Using bacterially expressed full-length and truncated proteins, we have shown that the catalytic domain is capable of phosphorylating itself. The association of KIPK with KCBP suggests regulation of KCBP or KCBP-associated proteins by phosphorylation and/or that KCBP is involved in targeting KIPK to its proper cellular location.