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Originally published In Press as doi:10.1074/mcp.M600188-MCP200 on August 3, 2006.
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Molecular & Cellular Proteomics 5:2124-2130, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Peptide Microarray Analysis of Substrate Specificity of the Transmembrane Ser/Thr Kinase KPI-2 Reveals Reactivity with Cystic Fibrosis Transmembrane Conductance Regulator and Phosphorylase*,S

Hong Wang and David L. Brautigan{ddagger}

From the Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. We have been interested in several examples of stable complexes formed between a protein kinase and a protein phosphatase. The kinases kinase/phosphatase/inhibitor-2 (KPI-2)1(1), Nek2 (NIMA-related kinase 2) (2, 3), and Aurora (4–6) are known to form complexes with protein phosphatase PP1. These complexes can perform as switches by mutual inactivation through phosphorylation and dephosphorylation and reversal by autophosphorylation and autodephosphorylation. We identified KPI-2 (also known as Lmr2, LMTK2, AATYK2, KIAA1079, Cprk, and BREK) as a kinase with an N terminus of two transmembrane segments followed by a kinase domain and a long cytoplasmic C-terminal tail. KPI-2 binds to PP1C through a canonical PP1 binding motif (VXF motif) in the C-terminal region. KPI-2 has a separate site for association with inhibitor-2 because inhibitor-2 binding was seen even when the VTF motif was mutated (to ATA) to eliminate PP1 binding. When we expressed the KPI-2 kinase domain it exhibited only Ser/Thr kinase activity during autophosphorylation or with the added substrate myelin basic protein (MBP). KPI-2 kinase domain phosphorylated PP1C at the Thr-320 site in the conserved PITPP motif, which attenuates PP1C activity (1).

KPI-2 is a member of human lemur (Lmr) kinase group named after the Madagascar primates with long tails because these membrane kinases have long C-terminal cytoplasmic tails. There are three members of this family, Lmr1 (KIAA0641, GenBankTM accession number AB014541) (7), Lmr2 (KIAA1079, GenBank accession number NM_014916) (1, 8, 9), and the more distant relative Lmr3 (KIAA1883, GenBank accession number AB067470) that are all classified as Tyr kinases in the human kinome tree based on their sequences (10). Lmr2 and Lmr3 are predicted transmembrane kinases, and Lmr1 lacks a transmembrane motif. The Lmr kinases were located in a branch closest to EGFR-HER and JAK-TYK Tyr kinases; however, Basic Local Alignment Search Tool (BLAST) analysis of the KPI-2 kinase domain gives highest scores for Trk kinases of neurotrophin receptors. Members of the Lmr group of kinases have also been called "apoptosis-associated protein Tyr kinases" (AATYK) (7, 11–17) because there is induction of AATYK1 (mouse homologue of Lmr1) in cerebellar granule cells induced to undergo apoptosis by low potassium levels, and overexpression of AATYK1 in these cells increased the number of apoptotic cells (12). The expression level of AATYK1 was increased in myeloid cells during growth arrest or differentiation (17). This family of kinases has not been found in zebrafish, Drosophila, or Caenorhabditis elegans, suggesting that they are exclusively mammalian kinases.

The Lmr2 kinase has been discovered independently by three approaches. We found KIAA1079 (Lmr2) as yeast two-hybrid prey from a mouse embryonic day 9.5 cDNA library using phosphatase inhibitor-2 as bait and demonstrated PP1 binding, so we named it KPI-2 (1). A separate yeast two-hybrid screen of a human brain cDNA library isolated KIAA1079 using the p35 subunit of CDK5 as bait. The kinase was named Cprk for cyclin-dependent kinase 5/p35-regulated kinase (8). The third approach used a database search for novel kinases in brain that revealed KIAA1079, and its mRNA was found primarily in brain by Northern hybridization and RT-PCR. This group therefore named KIAA1079 as brain-enriched protein kinase (BREK) (9). All three groups have reported autophosphorylation of the kinase, and either phosphoamino acid analysis or immunoblotting with phosphospecific antibodies showed only Ser(P) and Thr(P). This raised the issue that the biochemical properties were different from those predicted based on sequence similarities to various Tyr kinases. Co-expression of KIAA1079 with CDK5/p35 was reported to inhibit autophosphorylation possibly due to inhibitory CDK phosphorylation (8). Overexpressed BREK was phosphorylated upon stimulation of PC-12 cells with NGF, but it was not determined whether this was Tyr or Ser/Thr phosphorylation. Transfection of kinase-defective BREK enhanced neurite outgrowth in PC12 cells indicating that BREK might inhibit neurite outgrowth by phosphorylation (9). KIAA1079 is also called AATYK2 because its sequence is related to AATYK1 and AATYK3, but there are no reports about involvement of this kinase in apoptosis.

The cellular substrates of any of the Lmr kinases are unknown. To examine the specificity of KPI-2 kinase against a wide selection of possible peptide substrates we performed a PepChip kinase assay with an array of >1,100 peptides. We found phosphorylation of Ser and Thr but not Tyr peptides. Based on the peptide sequences we identified two unusual possible protein substrates, cystic fibrosis transmembrane conductance regulator (CFTR) (18) and glycogen phosphorylase (phos-b) (19). We show that these proteins are substrates for KPI-2 kinase, and phos-b was used as a highly specific KPI-2 substrate to demonstrate that NGF signaling in living cells reduces the activity of KPI-2 kinase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Constructs—
pCMV-KPI-2-Full length was constructed by inserting KPI-2 full length into pCMV C-terminal c-Myc-tagged vector, and pFastBac-His6-KPI-2 kinase was constructed by inserting KPI-2 fragment (residues 94–600) into pFastBac HTb vector as described before (1). This construct was chosen as kinase to include the minimal kinase fold (predicted as residues 137–407) plus an additional C-terminal segment that is unique to Lmr proteins and might be involved in regulation and/or substrate recognition. The solubility, stability, activity, and relative resistance to proteolytic pruning during purification suggest that this kinase has a globular native structure. A plasmid encoding the R domain of CFTR (residues 589–830) fused to GST was a kind gift from Dr. William Reenstra, University of Pennsylvania.

Expression and Purification of Recombinant KPI-2 Kinase Protein—
For protein expression, Sf9 cells were infected with recombinant baculoviruses, and cells were harvested 60 h later. The recombinant proteins were purified by IMAC as described before (1). The fractions containing KPI-2 kinase were pooled and loaded onto a HiTrapTM Q XL anion exchange column (Amersham Biosciences). Proteins were eluted by linear gradient of NaCl concentration from 0.2 to 0.7 M. Fractions were subjected to 9% SDS-PAGE and analyzed by GelCode Blue staining (Pierce). Fractions containing KPI-2 kinase were concentrated using Centricon YM-50 (Millipore) and then loaded to Ultragel AcA54 column (1.5 x 43 cm). Blue dextran 2000 was added to the loading sample to determine void volume of the column. The proteins were eluted at 10 ml/h with the buffer (20 mM Hepes, pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol, 0.1% ß-mercaptoethanol, 10% glycerol, 100 mM NaCl, and 20 µg/ml ovalbumin), and 80 fractions of 1 ml each were collected. The fractions containing protein were pooled, and the concentrations were measured by Bio-Rad protein assay.

PepChip Array Kinase Assay and Analysis—
PepChip kinase array was purchased from Pepscan Systems. The size of PepChip kinase slides is 25 x 75 mm. The full slide contains two duplicate arrays of 1,176 peptides each. For kinase assay, 50 µl of kinase reaction solution containing 500 ng/ml purified recombinant KPI-2 kinase (fractions 24 and 25 from AcA54, Fig. 1D, box), 2x kinase reaction buffer, 10 µM ATP, and 300 µCi/ml [33P]ATP) was added to the PepChip slide and incubated for 2 h at 30 °C. The 2x kinase reaction buffer includes 100 mM Hepes, pH 7.4, 40 mM MgCl2, 20% glycerol, 0.02 mg/ml BSA, and 0.02% Brij 35. After incubation, the slide was washed once with PBS-TX (1% Triton X-100, PBS), twice with NaCl-TX (1% Triton X-100, 2 M NaCl), and twice with distilled H2O and then air-dried. The phosphorylation was detected by PhosphorImager (GE Healthcare). The result was quantitated and analyzed by ImageQuant software.


Figure 1
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  		FIG. 1. Purification of recombinant KPI-2 kinase. The recombinant baculovirus of KPI-2 kinase (residues 94–600) was made using Bac-to-Bac baculovirus expression system. Recombinant proteins were purified by IMAC followed by ion exchange and size exclusion chromatography. A, constructs of KPI-2 used in this study. a, pCMV-KPI-2-Full length. TM stands for transmembrane domain. KD is kinase domain. VTF is PP1C binding motif. b, pFastBac-His6-KPI-2 kinase used in Sf9 cells. B, IMAC. The lysates from infected Sf9 were incubated with nickel-nitrilotriacetic acid resin for 2 h, and proteins were eluted by stepwise increase in concentration of imidazole-HCl (Im-HCl), pH 7, as indicated. C, fast protein liquid chromatography anion exchange chromatography. Fractions 5–10 from nickel-nitrilotriacetic acid column (B in box) were combined and loaded onto a HiTrap Q XL column. Proteins were eluted by linear gradient of NaCl concentration from 0.2 to 0.7 M. D, size exclusion chromatography. Fractions 12–25 from HiTrap Q XL column (C in box) were pooled and concentrated using Centricon YM-50. Blue dextran 2000 was added to the concentrated sample to determine void volume of column. The sample was loaded onto an Ultragel AcA54 column (1.5 x 43 cm), and 80 fractions of 1 ml each were collected. 10 µl of each fraction from above columns were subjected to 9% SDS-PAGE and analyzed by GelCode staining. The lower bands of D are ovalbumin added in the elution buffer to prevent loss of protein due to absorption to surfaces.

 
Cell Culture, Transfection, Immunoprecipitation, and Western Blot—
HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% newborn calf serum. Cells were transfected using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). After 48 h, the cells were incubated in the medium without serum for 3 h followed by treatment with 100 ng/ml NGF for 30 min. Cells were lysed with 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM ß-glycerophosphate, 1 mM dithiothreitol, 1 mM Na3VO4, 1 µM Microcystin-LR, plus protease inhibitors). For immunoprecipitation, equal amounts of proteins were incubated with anti-c-Myc-agarose (Sigma) for 2 h at 4 °C. The beads were washed three times with lysis buffer and once with kinase buffer and then subjected to SDS-PAGE for Western blot or used as kinase source for kinase assay.

KPI-2 Kinase Assays—
Kinase assays were performed by using either purified KPI-2 kinase or full-length KPI-2 kinase immunoprecipitated from HEK293T cells by c-Myc tag. KPI-2 kinases were incubated in a total reaction volume of 20 µl containing 10 mM MgCl2, 5 mg/ml different substrates, 100 µM [32P]ATP (10 µCi). For immunoprecipitated KPI-2 kinase, 200 µM Na3VO4, 20 nM Microcystin-LR, and 1 mM EGTA (final concentrations) were added to the kinase reaction. The phosphorylation was detected by PhosphorImager (GE Healthcare). The results were quantitated and analyzed by ImageQuant software.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Peptide Microarray Assay and Analysis—
The KPI-2 kinase domain (Fig. 1 A; residues 94–600) was produced in Sf9 cells by a baculovirus expression system and purified in three steps. First KPI-2 kinase was purified from the soluble extract by IMAC, and one major protein was recovered (Fig. 1B, box). This was the only radiolabeled protein observed during autophosphorylation with [32P]ATP (not shown). However, we suspected there were multiple kinases present at this level of purification because in kinase assays the preparation phosphorylated Ser/Thr residues in different substrates and also phosphorylated the synthetic polymer poly(Glu-Tyr). Proteins were loaded onto a HiTrap Q XL column and eluted by linear gradient of NaCl, and the KPI-2 kinase was pooled (Fig. 1C, box). Multiple peaks of MBP kinase were resolved (not shown), and at this stage of purification the KPI-2 kinase no longer exhibited any activity with poly(Glu-Tyr) as substrate, so we concluded that a contaminating protein Tyr kinase had been removed. For final and complete purification the KPI-2 was concentrated and loaded onto an Ultragel AcA54 column where it was separated from other proteins and eluted as a monomer (Fig. 1D, box) that was used as the purified kinase.

We incubated KPI-2 kinase with radioactive ATP and a PepChip microarray (Pepscan Systems), which contains duplicate sets of 1,152 different peptides with a median length of nine residues, based on known phosphorylation sites in the PhosphoBase database (phospho.elm.eu.org). Reaction conditions were optimized with a test array prior to assay with the complete array. The results in the two duplicate arrays on the same slide (Fig. 2) matched one another. We quantitated the radioactivity to get relative phosphorylation levels of the peptides with pixel intensities from a low of 1 to the highest at 48,113. The peptide phosphorylation was scored 0 to 5+ based on these pixel intensities (Table I). Of the 1,176 positions in the microarray (some peptides appear twice) a total of 74% were considered as negative (non-phosphorylated) compared with 36% as positive (phosphorylated). Of the 172 total peptides with tyrosine sites we detected a very low level of phosphorylation (pixel intensities, 1–8; score, 0–1) in five peptides. We attributed this activity to a trace contaminant of a Tyr kinase, most of which was removed during anion exchange chromatography. Of the positives only about 1% of the total peptides (14 of 1,152 total) showed strong phosphorylation +++ to +++++ (Table II). This attested to the purity and specificity of the KPI-2 kinase and the discrimination of the assay. No consensus sequence for substrate specificity could be discerned from the group of KPI-2 substrate peptides. However, the KPI-2 kinase did show preference for sites with basic residues, similar to PKA. KPI-2 also efficiently phosphorylated Ser/Thr residues adjacent to Pro as well as those not next to Pro, showing it has unique reactivity and substrate specificity distinct from PKA and other kinases.


Figure 2
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  		FIG. 2. PepChip KPI-2 kinase assay. A peptide microarray containing 2 x 1,176 peptides derived from known phosphorylation sites of kinases was assayed with KPI-2 kinase as described under "Experimental Procedures." The figure shows the duplicate array of peptides on one slide. The array is composed of a series of 7 x 7 spots in subarrays with four subarrays on the x axis and six subarrays on the y axis.

 

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  		TABLE I Summary of KPI-2 PepChip kinase array

The phosphorylation levels of peptides were quantitated with ImageQuant software. The lowest pixel value is 1, and the highest is 48,113. The values are divided into six groups, scored from 0 (–) to 5+. The numbers of peptides in each group were counted, and these results are summarized in the table.

 

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  		TABLE II Identity of the best KPI-2 peptide substrates

The 14 most intensely phosphorylated peptides scored from 3+ to 5+ (Table I) are listed. From left to right are peptide substrate name, score, position in the image from Fig 2, peptide sequence, and numbering of the phosphosite, if known, in the parent protein. MLRM, myosin regulatory light chain; H2B, histone 2B; KPBB, phosphorylase b kinase regulatory subunit ß; LBR, lamin-B receptor; Phos-b kinase, phosphorylase b kinase ß regulatory chain; NMZ1, N-methyl-D-aspartate receptor subunit NR1; TRIF, troponin I, fast skeletal muscle.

 
Our attention was drawn to the most intensely phosphorylated peptide (position X21 Y2; see Fig. 2 for peptide positions), which is derived from CFTR (20–22) and contains the Ser-737 phosphorylation site (Fig. 3 A). We identified four other peptides in the microarray that correspond to phosphorylation sites in CFTR. These peptides were much less intensely phosphorylated by KPI-2 kinase (Fig. 3A). We also noticed peptides corresponding to the single Ser phosphorylation site in phos-b (19, 23) (Fig. 3A). There were six different variants of the phos-b peptide in the array. The phosphorylation intensity of these peptides was not in the highest 1%, but we recognized phos-b as an example of a protein substrate that is exceptionally specific. There is only one kinase known to react with phos-b, namely phosphorylase b kinase (PhK), a Ca2+- and calmodulin-activated kinase (24–26). This raised a possibility for us to use phos-b as substrate to measure KPI-2 kinase activity in the presence of EGTA.


Figure 3
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  		FIG. 3. KPI-2 kinase assay with proteins. A, the substrates CFTR and phosphorylase b were identified from multiple separate peptides in the microarray. From left to right are peptide positions from the image in Fig. 2, relative intensities, peptide sequences, and the corresponding known kinases and phosphorylation sites. B, purified rabbit muscle phosphorylase b, purified recombinant GST-CFTR R domain (residues 589–830), and histone H1 were tested for phosphorylation by purified KPI-2 kinase. Kinase reaction was carried out by incubation of KPI-2 kinase with different substrates and [32P]ATP. PKA was used as a control kinase for comparison.

 
KPI-2 Phosphorylation of CFTR and Phosphorylase b—
To extend the results of the PepChip array assay, we performed KPI-2 kinase assays using different purified and recombinant proteins as substrates. These included recombinant GST-CFTR R domain (residues 589–830) expressed in bacteria, phos-b purified from rabbit muscle (27), and histone H1. As shown in Fig. 3B, all these substrates were phosphorylated by KPI-2 with histone H1 most intensely phosphorylated and phos-b and GST-CFTR phosphorylated about equally. GST was not phosphorylated as expected for the negative control. The results show that the peptide phosphorylation in the microarray assay was predictive of phosphorylation of the intact proteins. As an additional control the same protein substrates were incubated in parallel with PKA, a known kinase for CFTR (28). As shown in Fig. 3B, GST-CFTR was phosphorylated by PKA, consistent with previous reports that the R domain in CFTR has multiple sites of PKA phosphorylation (29, 30). Phosphorylation of GST-CFTR by PKA was more intense than the labeling of histone H1, whereas phos-b was not phosphorylated by PKA under the same conditions. This reinforced the conclusion that KPI-2 had different substrate specificity than PKA.

Assay of KPI-2 Kinase with Phosphorylase b as a Specific Substrate—
Our results indicated that we could use phos-b as specific substrate to measure KPI-2 kinase activity in reactions performed with EGTA to chelate calcium. KPI-2 has been implicated in NGF signaling in PC12 cells based on indirect evidence of cell shape changes in response to NGF after cells were stably transfected with active or inactive kinase (9). To directly test whether KPI-2 kinase activity changes in response to NGF we expressed full-length c-Myc-tagged KPI-2 in HEK293T cells, treated the cells with NGF, and compared them with an untreated control. KPI-2 was immunoprecipitated with anti-c-Myc antibodies, and a kinase assay was performed using [32P]ATP and phos-b as substrates. As shown in Fig. 4, phos-b was phosphorylated by KPI-2 kinase, and NGF treatment of the cells prior to immunoprecipitation reduced the KPI-2 kinase activity by more than 60% (p < 0.01, n = 5). This effect was dose-dependent on NGF (1–100 ng/ml). A similar reduction in KPI-2 activity with phos-b was seen upon addition of 10% fetal serum to cells (not shown). The results demonstrate that phos-b can be used as specific substrate of KPI-2 and show directly for the first time that NGF signaling inhibits KPI-2 kinase activity.


Figure 4
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  		FIG. 4. Use of phosphorylase b as specific substrate to measure KPI-2 kinase activity. HEK293T cells were transfected with full-length KPI-2 kinase using Lipofectamine 2000, and pcDNA3 vector was used as control. After 2 days, HEK293T cells were incubated without serum for 3 h and then treated with/without 100 ng/ml NGF for 30 min. KPI-2 kinase was immunoprecipitated using anti-c-Myc-agarose (Sigma). Kinase reaction was performed by incubating the agarose beads with phosphorylase b in the buffer containing MgCl2, [32P]ATP, and EGTA. The phosphorylation was detected by PhosphorImager, and phosphorylation levels were quantitated with ImageQuant Integration software. The graphs show the relative KPI-2 kinase activity (corrected for vector alone background activity) as the mean ± S.E. from five independent experiments (*, p < 0.01). The inset shows the PhosphorImager result from one experiment.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Among the 518 human protein kinases about 90 are classified as Tyr-specific based on sequence similarities in the catalytic domain (10). This study used a peptide microarray to test the substrate specificity of one such putative protein Tyr kinase of the Lmr branch of the kinase family, KPI-2 (1) (also known as AATYK2, Lmr2, LMTK2, Cprk, and BREK). Based on reaction with 1,152 peptides on the microarray only about 1% were phosphorylated effectively, and about one-third of the peptides showed some radiolabeling. These peptide substrates were based on a variety of sites of protein phosphorylation in the PhosphoBase database and had a mean length of nine residues to facilitate recognition. Contrary to its classification, KPI-2 did not phosphorylate Tyr-containing peptides but was specific for Ser/Thr peptides. This agrees with our previous results of KPI-2 autophosphorylation on Ser/Thr residues and a lack of reactivity with poly(Glu-Tyr) as a substrate. Thus, the Lmr group had been placed on a branch of the kinase family tree with the well known Tyr-specific kinases EGFR-HER and JAK-TYK (10) and is related by BLAST analysis to Trk Tyr kinase; however, the Lmr kinases need to be considered specific Ser/Thr kinases.

The specificity of KPI-2 for peptide substrates revealed reactivity with Pro-Ser and Pro-Thr sites in peptides from bovine MBP. Interestingly KPI-2 did not phosphorylate a number of other peptides for known phosphosites in MBP, including sites for mitogen-activated protein kinase (Thr-97), protein kinase C (Ser-45, Ser-114, and Ser-150), and PKA (Ser-10, Thr-33, Ser-54, Ser-109, Ser-131, and Ser-160). Phosphorylation of residues adjacent to Pro ((Ser/Thr)-Pro-X) is a hallmark of the so-called Pro-directed kinases (31, 32) such as CDK, glycogen synthase kinase-3, or mitogen-activated protein kinases. However, these kinases are different from KPI-2 in that they react with Ser/Thr followed by Pro, whereas KPI-2 phosphorylated Ser/Thr preceded by Pro. Yet KPI-2 also phosphorylated residues followed by Pro such as the PITPP site in protein phosphatase-1 (Thr-320) and the PRTP site in MBP. KPI-2 phosphorylation of Thr-320 in PP1 reduces the activity of the phosphatase and is removed by intramolecular autodephosphorylation. KPI-2 unlike other Pro-directed kinases is not exclusively specific for Pro sites. Many of the KPI-2 reactive sites had neighboring basic residues. In this way KPI-2 resembles PKA, which prefers (R/K)XXS as a substrate, but with both peptide microarrays and with proteins substrates these kinase are different from one another. Overall KPI-2 exhibited a unique pattern of substrate specificity as a Ser/Thr kinase.

Results from the peptide microarray led us to test two proteins as substrates for KPI-2. First, by far the best substrate in the entire array was a peptide corresponding to Ser-737 in the regulatory domain of CFTR. Indeed the R domain of CFTR is an excellent substrate for KPI-2. Both KPI-2 and CFTR are transmembrane proteins found in the plasma membrane of cells, and phosphorylation of CFTR regulates its channel activity (33–35). But it remains to be determined whether KPI-2 phosphorylates full-length CFTR in membranes and whether this has any functional consequence. The other protein identified from the PepChip array is phos-b, and we found that purified phos-b is indeed a substrate for KPI-2. Phos-b was the first kinase substrate ever discovered, by Fischer and Krebs (24) 50 years ago, and since that time no other kinase has been found capable of phosphorylating the single site at Ser-15 in the N terminus of the protein. To date the one and only phos-b kinase is a Ca2+- and calmodulin-dependent kinase composed of four subunits arranged in a tetramer of tetramers (24–26). Here we utilized a kinase assay including EGTA to exclude this phos-b kinase activity and effectively measured the activity of precipitated KPI-2. This assay was used to show that the kinase activity of KPI-2 was reduced by >60% in response to adding NGF to living cells. A previous report (9) showed the NGF stimulation of PC12 cells caused reduced mobility of KPI-2 in SDS-PAGE, consistent with NGF-induced phosphorylation. That response was blocked by chemical inhibitors K252a and chelerythrine. Together the results suggest that NGF stimulates phosphorylation of KPI-2, perhaps involving protein kinase C, which reduces its kinase activity. Other experiments have shown that CDK5 phosphorylation of KPI-2 reduced its autophosphorylation (8). Thus, KPI-2 seems to have multiple sites of phosphorylation that can inhibit its kinase activity. The availability of phos-b as a convenient and highly specific substrate for KPI-2 will facilitate future studies of the regulation of this unusual transmembrane kinase.


   ACKNOWLEDGMENTS
 
We thank Dr. William Reenstra, University of Pennsylvania, for the generous gift of pGEX-CFTR-R domain plasmid.


  FOOTNOTES
 
Received, May 19, 2006, and in revised form, July 31, 2006.

Published, MCP Papers in Press, August 3, 2006, DOI 10.1074/mcp.M600188-MCP200

1 The abbreviations used are: KPI-2, kinase/phosphatase/inhibitor-2; Lmr2 and LMTK2, lemur tyrosine kinase 2; Cprk, cyclin-dependent kinase 5/p35-regulated kinase; BREK, brain-enriched protein kinase; AATYK, apoptosis-associated protein-Tyr kinases; CFTR, cystic fibrosis transmembrane conductance regulator; phos-b, glycogen phosphorylase; PP1, protein phosphatase-1; NGF, nerve growth factor; EGFR-HER, epidermal growth factor receptor-human epidermal growth factor receptor; JAK, Janus kinase; HEK, human embryonic kidney; PKA, cAMP-dependent protein kinase; CDK, cyclin-dependent kinase. Back

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

{ddagger} To whom correspondence should be addressed. Tel.: 434-924-5892; Fax: 434-243-2829; E-mail: db8g{at}virginia.edu


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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