Differential expression of isoforms of the regulatory subunit of type II cAMP-dependent protein kinase in rat neurons, astrocytes, and oligodendrocytes

The RII-B isoform of the regulatory subunit (R) of cAMP-dependent protein kinase II is abundantly and selectively expressed in cerebral cortex (Erlichman, J., Sarkar, D., Fleischer, N., and Rubin, C. S. (1980) J. Biol. Chem. 255, 8179-8184). In contrast to the cytosolic RII-H isoform from heart and other non-neural tissues, a substantial fraction of cerebral cortex RII-B is tightly associated with cell organelles. In order to study the cellular basis for the localization and abundance of RII-B in this complex and heterogeneous tissue, rat cerebral cortex was fractionated into highly purified populations of neurons, astrocytes, and oligodendrocytes. In neurons and astrocytes more than 80% of the total cAMP-binding activity is contributed by RII subunits, whereas the myelin-producing oligodendrocytes contain nearly equal proportions of RI (from protein kinase I) and RII. Approximately 70% of RII and RI subunits are associated with the particulate fraction in each of the three types of brain cells. The nature of the RII isoforms expressed in the cytosolic and particulate fractions of the purified brain cells was established by performing Western immunoblot and indirect immunoprecipitation analyses with selective and sensitive polyclonal antibodies directed against RII-B. Astrocytes and neurons exhibit high levels of RII-B, whereas oligodendrocytes contain the RII-H isoform. Thus, the expression of RII isoforms is not uniform among brain cells that are anatomically and developmentally related. Rather, it appears that RII-B and RII-H are expressed in a cell-specific fashion within cerebral cortex and this might reflect an RII-mediated adaptation of protein kinase II to the specialized metabolic and functional roles of neurons, astrocytes, and oligodendrocytes.     

Differential binding of the regulatory subunits (RII) of cAMP-dependent protein kinase II from bovine brain and muscle to RII-binding proteins

The association of regulatory subunits (RII) of Type II cAMP-dependent protein kinase from bovine cerebral cortex (RII-B) and bovine cardiac and skeletal muscle (RII-H) with specific binding proteins in bovine brain cytosol and purified brain microtubules was demonstrated using a solid phase binding assay. RII-binding proteins present in bovine cerebral cortex were immobilized on nitrocellulose filters after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the filters with 32P-labeled regulatory subunits showed that both RII-B and RII-H interact with the 75,000-dalton calmodulin-binding protein (P75) and microtubule-associated protein 2 (MAP-2). However, significant differences in binding affinities and capacities were observed. RII-B displayed a higher affinity for P75 compared to RII-H while RII-H preferentially bound to MAP-2. Quantitation of radioactive RII bound to MAP-2 showed that MAP-2 bound 4-6 times more RII-H than RII-B. The differential binding affinities and capacities of RII-H and RII-B for MAP-2 were not affected by autophosphorylation since both phospho and dephospho forms of RII displayed the same binding characteristics. Competitive binding studies suggest that RII-H and RII-B bind to the same sites on MAP-2. The biochemical basis for the differential binding of RII-B and RII-H to the same sites of MAP-2 is unknown. However, other high affinity RII-binding proteins present in cerebral cortex (i.e. P75) might affect the affinity of RII-B for MAP-2. 32P-RI did not bind to P75 nor MAP-2 under the conditions used.     

Ca2+/Calmodulin-Dependent Protein Kinase II and Protein Kinase C Phosphorylate a Synthetic Peptide Corresponding to a Sequence That Is Specific for the γ2L Subunit of the GABAA Receptor

Abstract: The γ₂ subunit of the GABA_A receptor (GABA_A-R) is alternatively spliced. The long variant (γ_2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca²⁺/calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336–351 of the intracellular loop of the γ_2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The K _m values for PKC-and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 μM , respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the γ_2L subunit has been shown to be necessary for ethanol potentiation of the GABA_A-R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function.     

Amino acid sequence around a "hinge" region and its "autophosphorylation" site in bovine Lung cGMP-dependent protein kinase

An exposed "hinge" region of cGMP-dependent protein kinase is known to be susceptible to both limited proteolysis and autophosphorylation. A 91-residue fragment has been isolated from this region and its amino acid sequence has been compared with the analogous regions of the cAMP-dependent protein kinases. Although a resemblance among these sequences is not striking, the phosphorylation sites are in corresponding regions toward the NH2 termini, and there are indications of homology in the vicinity of their autophosphorylation sites. As in the cAMP-dependent protein kinase, the site of autophosphorylation and the site of susceptibility to limited proteolysis are very near each other in the primary structure. The actual site of autophosphorylation (the underlined threonine residue in Pro-Arg-Thr-Thr-Arg) is quite different from those in the regulatory subunit of Type II cAMP-dependent kinase or the site in Type I regulatory subunit that can be phosphorylated by the cGMP-dependent protein kinase.     

Monoclonal antibodies as structural probes of surface residues in the regulatory subunit of cAMP-dependent protein kinase II from porcine heart

Two murine monoclonal antibodies (H5 and B6) generated against bovine heart type II regulatory subunit of cAMP-dependent protein kinase were shown to cross-react equally well with the homologous subunit from porcine heart. The antibodies demonstrated specificity for only the type II regulatory subunit and showed negligible cross-reactivity with the type I regulatory subunit, the catalytic subunit, and cGMP-dependent protein kinase. Following limited proteolysis of type II regulatory subunit with chymotrypsin, the H5 monoclonal antibody was shown to cross-react with the Mr = 37,000 cAMP-binding domain corresponding to the COOH-terminal region of the polypeptide chain. To more specifically localize the antigenic sites, the porcine type II regulatory subunit was carboxymethylated and cleaved with cyanogen bromide. Both monoclonal antibodies cross-reacted with the NH2-terminal CNBr peptide, and this peptide demonstrated affinities similar to native bovine type II regulatory subunit in competitive displacement radioimmunoassays. Tryptic cleavage of this CNBr fragment destroyed all antigenicity for both monoclonal antibodies, whereas antigenicity was retained following chymotryptic digestion. A single major immunoreactive chymotryptic fragment that cross-reacted with H5 was isolated by gel filtration and reverse phase high performance liquid chromatography. this peptide retained the complete antigenic site and had the following sequence: Asn-Pro-Asp-Glu-Glu-Glu-Glu-Asp-Thr-Asp-Pro-Arg-Val-Ile-His-Pro-Lys-Thr-Asp-Gl n. This antigenic site was localized just beyond the major site of autophosphorylation, approximately a third of the distance from the NH2-terminal end of the polypeptide chain.     

High affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II-B. Cloning, characterization, and expression of cDNAs for rat brain P150

Cyclic AMP-dependent protein kinase II-B appears to be adapted for function in the mammalian central nervous system via the properties of its regulatory subunit (RII-B). RII-B is selectively expressed in the central nervous system, tightly associated with cerebral cortex membranes, and avidly complexed by the bovine brain calmodulin-binding protein designated P75 (Sarkar, D., Erlichman, J., and Rubin, C. S. (1984) J. Biol. Chem. 259, 9840-9846). Complexes of RII-B and P75 polypeptides can be purified to near homogeneity from either membrane or cytosolic fractions of brain homogenates, suggesting that the binding protein plays a role in determining the central nervous system-specific properties of protein kinase II-B. To investigate the properties of a prototypic, nonabundant, RII-B-binding protein, we have cloned and characterized cDNAs for rat brain P150, a homolog of bovine brain P75. cDNAs were retrieved from a lambda gt11 expression library using 32P-labeled RII-B as a functional probe. cDNA inserts (800 and 1100 base pairs) subcloned into expression plasmids directed the production of partial P150 polypeptides in Escherichia coli that bind RII-B. Sequence analyses disclosed that P150 is a previously uncharacterized protein that contains multiple octapeptide repeats as well as unique sequences. Antibodies directed against 15-residue peptides corresponding to either repeated or unique sequences bound the polypeptides expressed in E. coli and a 150-kDa protein in rat brain membranes and cytosol. Moreover, the immunoprecipitated 150-kDa protein exhibited high affinity RII-B-binding activity. Finally, 3' deletion analysis demonstrated that a 15-amino acid segment of P150 is essential for binding with RII-B.     

Characterization of a fluorescent substrate for the adenosine 3',5'-cyclic monophosphate-dependent protein kinase

A synthetic tetradecapeptide derived from the phosphorylation site of the beta-subunit of phosphorylase kinase (Arg-Thr-Lys-Arg-Ser-Gly-Ser-Val-Tyr-Glu-Pro-Leu-Lys-Ile) is a highly efficient substrate for the cAMP-dependent protein kinase, exhibiting a 36% decrease in the intrinsic tyrosine fluorescence on phosphorylation. The fluorescence changes in continuous assays were monitored to demonstrate the roles of protein kinase effectors (cAMP, the type II regulatory subunit, and the 8000-Da heat-stable inhibitor) in the regulation of the enzyme and to determine Km and Vmax. The phosphorylation reaction requires 1 mol ATP/mol peptide. Amino acid analysis demonstrates the presence of phosphoserine in the phosphorylated peptide. Auxiliary experiments show that tyrosine phosphorylation can also be detected fluorometrically and distinguished from serine or threonine phosphorylation.     

Phosphorylation of synthetic peptide analogs of rabbit cardiac troponin inhibitory subunit by the cyclic AMP-dependent protein kinase.

A series of synthetic peptide analogs of the cardiac troponin inhibitory subunit (TN-1) phosphorylation site sequence, Arg12-Pro-Ala-Pro-Ala-Val-Arg18-Arg19-Ser20-Asp21-Arg22-Ala, have been tested as substrates for the catalytic subunit of the cyclic AMP-dependent protein kinase (EC 2.7.1.37, ATP:protein phosphotransferase). As substrates, these peptides were generally inferior to the pyruvate kinase analog peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly or its COOH-terminal amide analog. Replacing Arg-19 with alanine had only a minor effect on the kinetics of phosphorylation of the TN-1 peptide analog. In contrast, replacement of Arg-22 and Arg-18 with alanine resulted in marked enhancement and reduction of the Vmax, respectively. The results of this study have demonstrated that synthetic peptide analogs of the local phosphorylation site sequences of natural substrates may differ widely in their capacity to act as substrates for this protein kinase. In the case of the TN-1 peptide analogs, the contribution of the 4 arginine residues can be distinguished in terms of their influence on the kinetics of phosphorylation.     

The 44-kDa Regulatory Subunit of the Paramecium cAMP-Dependent Protein Kinase Lacks a Dimerization Domain and May Have a Unique Autophosphorylation Site Sequence

ABSTRACT. The 44-kDa regulatory subunit (R₄₄) of one form of cAMP-dependent protein kinase of Paramecium was purified, and two partial internal amino acid sequences from it were used to clone the corresponding cDNA. This R₄₄ cDNA clone was 1022-bp long, including 978 bp of coding sequence and 7 bp and 37 bp of 5' and 3' untranslated sequences, respectively. A 1.1-kb mRNA was labeled on a Northern blot. The deduced R₄₄ amino acid sequence had 31%–38% positional identity to the sequences of other cloned cAMP-dependent protein kinase regulatory subunits. R₄₄ sequence showed equal sequence similarity to mammalian types I and II regulatory subunits. The N -terminal sequence encoding the regulatory subunit dimerization domain found in most regulatory subunits is not present in the R₄₄ clone, confirming the lack of regulatory subunit dimer formation previously reported for the Paramecium cAMP-dependent protein kinase. The putative autophosphorylation site of R₄₄ contains the amino acid sequence TRTS, distinct from the consensus sequence RRXS, where X is any residue, found in other autophosphorylated cAMP-dependent protein kinase regulatory subunits and many cAMP-dependent protein kinase substrates.     

Formation of protein kinase recognition sites by covalent modification of the substrate. Molecular mechanism for the synergistic action of casein kinase II and glycogen synthase kinase 3

The mechanism for synergistic phosphorylation by glycogen synthase kinase 3 (GSK-3) and casein kinase II was studied using a synthetic peptide which contains the sequence of a potentially important proline/serine-rich regulatory region of rabbit muscle glycogen synthase. The peptide, Ac-PRPAS(3a)VPPS(3b)PSLS(3c)RHSS(4)PHQS(5) EDEEEP-amide, has five known phosphorylation sites of the native enzyme designated sites 3a, 3b, 3c, 4, and 5, which are spaced every fourth residue. The peptide was phosphorylated specifically at site 5 by casein kinase II with an apparent Km of 23 microM, but it was not phosphorylated by GSK-3. However, after initial phosphorylation of site 5 by casein kinase II, the peptide became an effective substrate for GSK-3 with an apparent Km of 2 microM. GSK-3 introduced up to four phosphates and appeared to catalyze the sequential modification of sites 4, 3c, 3b, and 3a, respectively. The results can be explained if GSK-3 recognizes the sequence -SXXXS(P). Phosphorylation of site 5 by casein kinase II creates this recognition site. Thereafter, each successive phosphorylation introduced by GSK-3 generates a new recognition site. The results provide a molecular basis to explain the synergistic action of casein kinase II and GSK-3 that is also observed with native glycogen synthase. In addition, this investigation emphasizes how protein recognition sites in some cellular targets may have to be formed post-translationally.     

The 90-kilodalton peptide of the heme-regulated eIF-2 alpha kinase has sequence similarity with the 90-kilodalton heat shock protein

Highly purified preparations of the heme-controlled eIF-2 alpha (eukaryotic peptide initiation factor 2 alpha subunit) kinase of rabbit reticulocytes contain an abundant 90-kilodalton (kDa) peptide that is immunologically cross-reactive with spectrin and that modulates the activity of the enzyme [Kudlicki, W., Fullilove, S., Read, R., Kramer, G., & Hardesty, B. (1987) J. Biol. Chem. 262, 9695-9701]. The amino-terminal sequence of the 90-kDa protein has a high degree of similarity with the known amino-terminal sequences of the Drosophila 83-kDa heat shock protein (20 out of 22 residues) and with other related heat shock proteins. The amino acid sequence of a tryptic phosphopeptide isolated by high-performance liquid chromatography from the eIF-2 alpha kinase associated 90-kDa protein after phosphorylation by casein kinase II is shown to be identical with a 14 amino acid segment of the known sequence of the Drosophila 83-kDa heat shock protein. Results of hydrodynamic studies indicate a highly elongated structure for the reticulocyte protein, characteristic of a structural protein. Additional structural similarities between the eukaryotic heat shock proteins, the reticulocyte eIF-2 alpha kinase associated 90-kDa peptide, and spectrin are discussed.     

Sequence of two phosphorylated sites in the catalytic subunit of bovine cardiac muscle adenosine 3':5'-monophosphate-dependent protein kinase.

The sequences of two phosphopeptides isolated from the catalytic subunit of bovine cardiac muscle cAMP-dependent protein kinase (type II) and from two of its cyanogen bromide fragments, have been determined. One phosphorylation site is a threonyl residue located approximately 180 residues from the blocked NH2 terminus. Its sequence is: -Gly-Arg-Thr-Trp-Thr(P)-Leu-Cys- and includes one of the three sulfhydryl groups present in the molecule. The second phosphorylated site within the sequence: -Val-Ser(P)-Ile-Asn- is located towards the carboxyl end of the protein where the other 2 cysteinyl residues also reside. The finding that phosphorylation of the catalytic subunit occurs on two discrete sites rather than at random suggests that it might be of physiological importance, e.g. in the regulation of enzyme activity.     

Serine-23 Is a Major Protein Kinase A Phosphorylation Site on the Amino-Terminal Head Domain of the Middle Molecular Mass Subunit of Neurofilament Proteins

Abstract : We have shown previously that phosphate groups on the amino-terminal head domain region of the middle molecular mass subunit of neurofilament proteins (NF-M) are added by second messenger-dependent protein kinases. Here, we have identified Ser²³ as a specific protein kinase A phosphorylation site on the native NF-M subunit and on two synthetic peptides, S₁ (¹⁴RRVPTETRSSF²⁴) and S₂ (²¹RSSFSRVSGSPSSGFRSQSWS⁴¹), localized within the amino-terminal head domain region. Ser²³ was identified as a phosphorylation site on the ³²P-labeled α-chymotryptic peptide that carried >80% of the ³²P-phosphates incorporated into the NF-M subunit by protein kinase A. The synthetic peptides S₁ and S₂ were phosphorylated 18 and two times more efficiently by protein kinase A than protein kinase C, respectively. Neither of the peptides was phosphorylated by casein kinase II. The sequence analyses of the chemically modified phosphorylated serine residues showed that Ser²³ was the major site of phosphorylation for protein kinase A on both S₁ and S₂ peptides. Low levels of incorporation of ³²P-phosphates into Ser²², Ser²⁸, and Ser³² by protein kinase A were also observed. Protein kinase C incorporated ³²P-phosphates into Ser²², Ser²³, Ser²⁵, Ser²⁸, Ser³², and a threonine residue, but none of these sites could be assigned as a major site of phosphorylation. Analyses of the phosphorylated synthetic peptides by liquid chromatography-tandem mass spectrometry also showed that protein kinase A phosphorylated only one site on peptide S₁ and that ions with up to four phosphates were detected on peptide S₂. Analysis of the data from the tandem ion trap mass spectrometry by using the computer program PEPSEARCH did not unequivocally identify the specific sites of phosphorylation on these serine-rich peptides. Our data suggest that Ser²³ is a major protein kinase A-specific phosphorylation site on the amino-terminal head region of the NF-M subunit. Phosphorylation of Ser²³ on the NF-M subunit by protein kinase A may play a regulatory role in neurofilament assembly and/or the organization of neurofilaments in the axon.     

Phosphorylation of calcineurin: effect on calmodulin binding

The effect of phosphorylation of calcineurin on calmodulin (CaM) binding was examined using a synthetic peptide which contains the CaM-binding domain and the serine phosphorylation site. The peptide, corresponding to residues 391-414 of brain calcineurin A subunit, was rapidly phosphorylated by protein kinase C and Ca2+/CaM-dependent protein kinase II but not by cAMP-dependent protein kinase. Phosphorylation of peptide 391-414 did not significantly alter the binding of CaM when compared to the non-phosphorylated peptide.     

Phosphorylation of smooth muscle myosin light chain kinase by Ca2+/calmodulin-dependent protein kinase II: comparative study of the phosphorylation sites

Smooth muscle myosin light chain kinase (MLC-kinase) was rapidly phosphorylated in vitro by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) to a molar stoichiometry of 2.77 +/- 0.15 associated with a threefold increase in the concentration of calmodulin (CaM) required for half-maximal activation of MLC-kinase. Binding of CaM to MLC-kinase markedly reduced the phosphorylation stoichiometry to 0.21 +/- 0.05 and almost completely inhibited phosphorylation of sites in two peptides (32P-peptides P1 and P2) with reduced phosphorylation of peptide P3. By analogy, cAMP-dependent protein kinase phosphorylated MLC-kinase to a stoichiometry of 3.0 or greater in the absence of CaM with about a threefold decrease in the apparent affinity of MLC-kinase for CaM. Binding of CaM to MLC-kinase inhibited the phosphorylation to 0.84 +/- 0.13. Complete tryptic digests contained two major 32P-peptides as reported previously. One of the peptides, whose phosphorylation was inhibited in the presence of excess calmodulin, appeared to be the same as P2. Automated Edman sequence analysis suggested that both CaM-kinase II and cAMP-dependent protein kinase phosphorylated this peptide at the second of the two adjacent serine residues located at the C-terminal boundary of the CaM-binding domain. However, the other peptide phosphorylated by cAMP-dependent protein kinase, regardless of whether CaM was bound, was different from P1 and P3. Thus, MLC-kinase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of CaM-kinase II and is blocked by Ca2+/CaM-binding.     

Sequence determinants on the NR2A and NR2B subunits of NMDA receptor responsible for specificity of phosphorylation by CaMKII

Calcium/calmodulin-dependent protein kinase type II (CaMKII) and NMDA-type glutamate receptor (NMDAR) are neuronal proteins involved in learning and memory. CaMKII binds to the NR2B subunit of NMDAR in more than one mode, a stable association involving a noncatalytic site on CaMKII and an enzyme-substrate mode of interaction by its catalytic site. The latter binding results in phosphorylation of serine-1303 on NR2B. We have investigated this binding by studying the kinetics of phosphorylation of synthetic peptides harboring nested sequences of the phosphorylation site motif. We find that residues 1292-1297 of NR2B enhance the affinity of the catalytic site-mediated binding of CaMKII to the minimal phosphorylation site motif, 1298-1308 of NR2B, as evident from measurements of K(m) values for phosphorylation. However, CaMKII shows decreased affinity towards the closely related NR2A subunit due to an -Ile-Asn- motif present as a natural insertion in the analogous sequence on NR2A.     

CK2-mediated phosphorylation of a type II regulatory subunit of cAMP-dependent protein kinase from the mollusk Mytilus galloprovincialis

Two isoforms of regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), were identified so far in the sea mussel Mytilus galloprovincialis. Out of them, only R(myt2) was phosphorylated in vitro by casein kinase 2 (CK2) using GTP as phosphate donor. CK2 catalytic subunit (CK2alpha) itself was sufficient to phosphorylate R(myt2), but phosphorylation was enhanced by the presence of the regulatory subunit CK2beta. Even in the absence of CK2, R(myt2) was phosphorylated to a certain extent when it was incubated with GTP. This basal phosphorylation was partially abolished by the known inhibitors apigenin and emodin, which suggests the presence of a residual amount of endogenous CK2 in the preparation of purified R subunit. CK2-mediated phosphorylation significantly decreases the ability of R(myt2) to inhibit PKA catalytic (C) subunit activity in the absence of cAMP. On the other hand, the sequence of several peptides obtained from the tryptic digestion of R(myt2) showed that mussel protein contains the signature sequence common to all PKA family members, within the "phosphate binding cassette" (PBC) A and B. Moreover, the degree of identity between the sequences of peptides from R(myt2), as a whole, and those from type II R subunits was 68-75%, but the global identity percentage with type I R subunits was only about 30%, so that R(myt2) can be classified as a type II R subunit.     

Phosphorylation of high mobility group protein 14 by casein kinase II

Phosphorylation of chromosomal high mobility group (HMG) protein 14 by casein kinase II has been characterized. Two mol of 32P are incorporated per mol of bovine HMG 14. Kinetic analysis provided evidence for two distinct sites with apparent Km values of 14.5 and 134 microM and respective Vmax values of 0.17 and 0.68 mumol/min/mg casein kinase II. Tryptic peptide mapping identified two phosphorylated products, each with phosphoserine. Amino acid composition and sequence analysis demonstrate that the major high affinity phosphorylation site for casein kinase II is serine 89. This sequence located at the carboxyl-terminal of HMG 14 contains the primary sequence determinants for casein kinase II. On the basis of reverse-phase high performance liquid chromatography and amino acid analysis, HMG 14, serine 99 represents the low affinity phosphorylation site.     

Phosphorylation of serine-15 of maize leaf sucrose synthase. Occurrence in vivo and possible regulatory significance.

Experiments were conducted to determine whether sucrose synthase (SuSy) was phosphorylated in the elongation zone of maize (Zea mays L.) leaves. The approximately 90-kD subunit of SuSy was 32P-labeled on seryl residue(s) when excised shoots were fed [32P]orthophosphate. Both isoforms of SuSy (the SS1 and SS2 proteins) were phosphorylated in vivo, and tryptic peptide-mapping analysis suggested a single, similar phosphorylation site in both proteins. A combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and automated Edman sequencing analysis unequivocally identified the phosphorylation site in the maize SS2 protein as serine-15. This site was phosphorylated in vitro by endogenous protein kinase(s) in a strictly Ca(2+)-dependent manner. A synthetic peptide, based on the phosphorylation site sequence, was used to identify and partially purify an endogenous Ca(2+)-dependent protein kinase(s) from the maize leaf elongation zone and expanding spinach leaves. Phosphorylation of SuSy in vitro selectively activates the cleavage reaction by increasing the apparent affinity of the enzyme for sucrose and UDP, suggesting that phosphorylation may be of regulatory significance. Conservation of the phosphorylation site, and the sequences surrounding it, among plant species suggests that phosphorylation of SuSy may be widespread, if not universal, in plants.