Abstract: Cellular responses to many hormones and neurotransmitters wane rapidly despite continuous exposure of cells to these stimuli. This phenomenon, termed desensitization, has been particularly well studied for the stimulation of cAMP levels by plasma membrane beta-adrenergic receptors (beta AR). The molecular mechanisms underlying rapid beta AR desensitization do not appear to require internalization of the receptors, but rather an alteration in the functioning of beta AR themselves that uncouples the receptors from the stimulatory G protein Gs. This uncoupling phenomenon involves phosphorylation of beta AR by at least two kinases, PKA and the beta AR kinase (beta ARK), which are activated under different desensitizing conditions. Receptor phosphorylation by the two kinases leads to desensitization of the receptor response via distinct biochemical mechanisms, and additional cytosolic factors appear to be involved in the case of beta ARK. Numerous experimental approaches have been used recently to elucidate th...

Abstract: MAP kinase (relative molecular mass, 42,000), a low abundance serine--threonine protein kinase, is transiently activated in many cell types by a variety of mitogens, including insulin, epidermal growth factor, and phorbol esters. In vitro, MAP kinase will phosphorylate and reactivate S6 kinase II previously inactivated by phosphatase treatment. Because many of the stimuli that activate MAP kinase are also stimulators of cell proliferation, and regulation of the cell cycle seems to involve a network of protein kinases, MAP kinase could be important in the transmission of stimuli eventually leading to the progression from G0 to G1 in the cell cycle. Activated MAP kinase contains both phosphotyrosine and phosphothreonine. We report here that MAP kinase can be deactivated completely by treatment with either phosphatase 2A, a protein phosphatase specific for phosphoserine and phosphothreonine, or CD45, a phosphotyrosine-specific protein phosphatase. We demonstrate that MAP kinase is only active when both tyrosyl and threonyl residues are phosphorylated and suggest therefore that the enzyme functions in vivo to integrate signals from two distinct transduction pathways.

Abstract: Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a approximately 46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communication-competent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.

Abstract: Phospholipase C-gamma 1 (PLC-gamma 1), an isozyme of the phosphoinositide-specific phospholipase C family, which occupies a central role in hormonal signal transduction pathways, is an excellent substrate for the epidermal growth factor (EGF) receptor tyrosine kinase. Epidermal growth factor elicits tyrosine phosphorylation of PLC-gamma 1 and phosphatidylinositol 4,5-bisphosphate hydrolysis in various cell lines. The ability of tyrosine phosphorylation to activate the catalytic activity of PLC-gamma 1 was tested. Tyrosine phosphorylation in intact cells or in vitro increased the catalytic activity of PLC-gamma 1. Also, treatment of EGF-activated PLC-gamma 1 with a tyrosine-specific phosphatase substantially decreased the catalytic activity of PLC-gamma 1. These results suggest that the EGF-stimulated formation of inositol 1,4,5-trisphosphate and diacylglycerol in intact cells results, at least in part, from catalytic activation of PLC-gamma 1 through tyrosine phosphorylation.

Abstract: Activation of the T-cell antigen receptor (TCR) results in tyrosine phosphorylation of the TCR zeta chain and other intracellular substrates. Two other T-cell integral membrane proteins, CD4 and CD8, are associated with the protein-tyrosine kinase (PTK), lck. Despite evidence that activation of this enzyme results in TCR-zeta chain phosphorylation, it has not been shown that the TCR activates lck. We have sought evidence that the TCR is associated with a PTK. In this study we use digitonin to solubilize a murine T-cell hybridoma and demonstrate that antibodies binding extracellular but not intracellular domains of the TCR specifically coprecipitate only the fyn PTK and not lck or yes, two other kinases found in these cells. The association of the fyn PTK with the TCR might enable the T cell to independently regulate two PTKs through surface receptors.

Abstract: The turnover and processing of the Alzheimer beta/A4 amyloid precursor protein (beta APP) has been studied in PC12 cells after treatment with agents that regulate protein phosphorylation. Phorbol 12,13-dibutyrate, an agent that stimulates protein kinase C, decreased the levels of mature beta APP and increased the levels of 15- and 19-kDa peptides. These peptides appeared to be COOH-terminal fragments of beta APP, which arose when phorbol 12,13-dibutyrate increased the rate of proteolytic processing of mature forms of beta APP. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, also led to decreased levels of mature beta APP and increased levels of the 15- and 19-kDa peptides. H-7, an inhibitor of protein kinase C and of several other protein kinases, apparently decreased the rate of proteolytic processing of mature beta APP. The sizes of the putative COOH-terminal fragments observed after treatment with either phorbol 12,13-dibutyrate or okadaic acid suggest that one or both may contain the entire beta/A4 region of beta APP and thus be amyloidogenic. Our results support the hypothesis that abnormal protein phosphorylation may play a role in the development of the cerebral amyloidosis that accompanies Alzheimer disease.

Abstract: Triggering of the antigen-specific T cell receptor-CD3 complex (TCR-CD3) stimulates a rapid phospholipase C-mediated hydrolysis of inositol phospholipids, resulting in the production of second messengers and in T cell activation and proliferation. The role of tyrosine phosphorylation in these events was investigated with a tyrosine protein kinase (TPK) inhibitor, genistein. At doses that inhibited TPK activity and tyrosine phosphorylation of the TCR zeta subunit, but not phospholipase C activity, genistein prevented TCR-CD3-mediated phospholipase C activation, interleukin-2 receptor expression, and T cell proliferation. These findings indicate that tyrosine phosphorylation is an early and critical event that most likely precedes, and is a prerequisite for, inositol phospholipid breakdown during receptor-mediated T cell activation.

Abstract: Antiphosphotyrosine immunoblots were used to characterize tyrosine phosphorylated proteins after stimulation of the human TCR Increased tyrosine phosphorylation was evident on at least 12 substrates within 2 min after ligation of the TCR with mAb Analysis of the time course for increased tyrosine phosphorylation revealed distinct patterns Increased phosphorylation of 135-kDa and 100-kDa substrates was evident within 5 s, whereas increased phosphorylation of the TCR-zeta-chain required several minutes after treatment with anti-CD3 mAb This rapid cellular tyrosine phosphorylation occurred independent of the cell cycle, as it occurred after stimulation of resting T cells, T cell blasts, and the Jurkat T cell leukemia line When the TCR complex was cross-linked together with the CD4 receptor by heteroconjugate anti-CD3/CD4 mAb, an increased magnitude of tyrosine phosphorylation occurred, although no new substrates could be detected The increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates was specific in that anti-HLA class I, anti-CD6, anti-CD7, and anti-CD28 antibodies did not cause increased tyrosine phosphorylation Anti-CD4 stimulation of resting T cells did not cause increased tyrosine phosphorylation of pp100 and pp135, suggesting that the CD4-associated kinase, lck, does not account for the tyrosine phosphorylation observed after TCR stimulation Similarly, pharmacologic treatment of cells with phorbol ester and calcium ionophore did not cause increased tyrosine phosphorylation of these substrates, indicating that activation of protein kinase C or phospholipase C does not account for these early increases in tyrosine phosphorylation The time of onset of pp100 phosphorylation, and the magnitude of phosphorylation correlated with the magnitude of calcium mobilization when cells were stimulated with different forms of TCR stimulation When cells were labeled with [3H]myoinositol and analyzed after stimulation by anti-CD3 mAb, increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates preceded the activation of phospholipase C, as measured by the appearance of inositol 1,4,5-trisphosphate This occurred in both T cell blasts and in the Jurkat T cell line Thus, these findings show that increased tyrosine phosphorylation is the earliest yet detected signal observed after ligation of the TCR complex, and furthermore suggest that tyrosine phosphorylation might link the TCR to the phosphatidylinositolbisphosphate hydrolysis signaling pathway

Abstract: The c-Myb nuclear oncoprotein is phosphorylated in vitro and in vivo at an N-terminal site near its DNA-binding domain by casein kinase II (CK-II) or a CK-II-like activity. This in vitro phosphorylation reversibly inhibits the sequence-specific binding of c-Myb to DNA. The site of this phosphorylation is deleted in nearly all oncogenically activated Myb proteins, resulting in DNA-binding that is independent of CK-II. Because CK-II activity is modulated by growth factors, loss of the site could uncouple c-Myb from its normal physiological regulator.

Abstract: SIGNALLING by membrane immunoglobulin, the B-lymphocyte antigen receptor, regulates B-cell maturation and activation. Crosslinking of membrane immunoglobulin by antigen or by anti-immunoglobulin antibodies inactivates immature B cells, eliminating many of the B cells capable of producing auto-antibodies1. By contrast, crosslinking of membrane immunoglobulin promotes activation of mature B cells for clonal expansion and antibody production against foreign antigens2. Crosslinking membrane IgM on the immature B-cell line WEHI-231 induces growth arrest3. This response may be analogous to the deletion or inactivation of immature B cells that is induced by antigen or anti-IgM antibodies. Membrane immunoglobulin crosslinking stimulates phosphoinosi-tide hydrolysis, which leads to increases in intracellular calcium and activation of protein kinase C4–6. The induced phosphoinositide breakdown is important for inhibiting WEHI-231 growth (ref. 7 and D. Page, M.R.G., K. Fahey, L. Matsuuchi and A.L.D., manuscript submitted for publication), but may not be sufficient, as agents that elevate calcium and activate protein kinase C cause only partial growth arrest7. We now show that in both mature splenic B cells and the immature B–cell line WEHI-231 crosslinking membrane immunoglobulin also stimulates phosphorylation of protein tyrosine, a reaction that has been implicated as a key regulator of cell growth. Most of these phosphorylations were not a consequence of the phosphoinositide pathway. Thus, tyrosine phosphorylation is a second mode of transmembrane signalling by membrane immunoglobulin.

Abstract: Some genital human papillomavirus (HPV) types, such as 16 and 18, are highly associated with malignant cervical tumors while others, such as HPV 6, are only rarely found in these malignancies. The E7 oncoproteins of HPV 6, 16 and 18 each have a 17 amino acid region with striking homology to adenovirus E1a and SV40 LT. E1a, LT and the E7 oncoprotein of HPV16 all bind the cellular Rb protein in vitro, and for E1a and LT this region of homology contains sequences essential for interaction with Rb. We have now found that in HPV 16 E7 this region (amino acids 21-37) contains two separate biochemical activities, each of which contributes to E7-mediated transformation. Rb binding was localized to the N terminus of this region, while the C terminus was shown to serve as a substrate for casein kinase (CK) II, which phosphorylated serine-31 and serine-32. Replacement of the two serines by non-phosphorylatable amino acids led to a reduction in transforming activity and abolished phosphorylation but did not affect Rb binding. Rb binding and CK II phosphorylation were also examined for the E7 proteins of HPV 6 and HPV 18. HPV 16 and 18 E7 bound similar amounts of Rb, but HPV 6 E7 consistently bound less. Phosphorylation rates also varied, with HPV 18 E7 being 2-fold faster than HPV 16 E7, which in turn was 2-fold faster than HPV 6 E7. We conclude that Rb binding and phosphorylation of E7 by CKII are independent activities which are required for efficient transformation by E7 and that these activities correlate directly with the relative oncogenic potential of these viruses.

Abstract: The microtubule array in neuronal cells undergoes extensive growth, dynamics and rearrangements during neurite outgrowth While little is known about how these changes are regulated, microtubule-associated proteins (MAPs) including tau protein are likely to perform an important role Tau is one of the MAPs in mammalian brain When isolated it is usually a mixture of several isoforms containing between 341 and 441 residues that arise from alternative splicing Tau can be phosphorylated by several protein kinases Phosphorylation at certain sites results in major structural and functional changes, as seen by changes in electrophoretic mobility, interaction with microtubules, molecular length and elasticity Here we show that the sites of phosphorylation by four kinases (PKA, PKC, CK and CaMK) all lie in the C-terminal microtubule-binding half of tau, but only the phosphorylation by CaM kinase shows the pronounced shift in electrophoretic mobility characteristic for tau from Alzheimer neurofibrillary tangles By using a combination of limited proteolysis, protein sequencing and protein engineering we show that a single phosphorylation site is responsible for this shift, located at Ser 405 in the C-terminal tail of the protein outside the region of internal repeats Phosphorylation at this site not only reduces the electrophoretic mobility of tau, it also makes the protein long and stiff, as shown earlier The site is likely to be phosphorylated in tau from Alzheimer neurofibrillary tangles

Abstract: The effect of phosphorylation on the functional activity of eukaryotic elongation factor 2 (eEF-2) was studied using a purified phosphorylated factor. The modified factor was unable to stimulate protein synthesis in an eEF-2-dependent rabbit reticulocyte lysate. The functional alteration was further analyzed by measuring the effects of phosphorylation on the ability of the factor to catalyse the ribosome-dependent hydrolysis of GTP. Kinetic analysis showed that both phosphorylated and unmodified factor was able to hydrolyse GTP with approximately the same maximum rate, indicating that the rate of nucleotide exchange was not impaired by the modification. However, the phosphorylated factor showed a marked reduction in the second-order rate constant, suggesting that the phosphorylation interfered with ribosome · eEF-2 complex formation by reducing the affinity of eEF-2 for the ribosome. This assumption was confirmed by direct measurements of the dissociation constants for the ribosomal complexes containing unmodified and phosphorylated eEF-2.

Abstract: A DNA-activated protein kinase (DNA-PK) was purified from nuclei of HeLa cells. Activity was associated with a single high-molecular-mass (approximately-300,000 Da) polypeptide when analyzed by gel filtration, denaturing polyacrylamide gel electrophoresis, and Western immunoblotting using a monoclonal antibody that also inhibits enzyme activity. Nuclear localization was indicated by subcellular fractionation and confirmed by immunofluorescence on whole cells. Double-stranded DNA stimulated phosphorylation of the 300-kDa polypeptide in purified preparations as well as phosphorylation of the exogenous substrates alpha-casein, simian virus 40 large T antigen, and the human heat shock protein hsp90. Autophosphorylation led to inactivation of the enzyme. The phosphorylation of casein was stimulated over 30-fold by DNA and was specific for serine and threonine residues. Bovine serum albumin and histone H1 were poor substrates for DNA-PK, and no phosphorylation of immunoglobulin G or histones other than H1 was observed. Supercoiled or heat-denatured DNA and synthetic double-stranded RNA or RNA-DNA copolymers did not stimulate casein phosphorylation by DNA-PK. Interaction of the enzyme with DNA in the absence of exogenous substrates was demonstrated by thermal inactivation and gel mobility shifts. These characteristics identify DNA-PK as distinct from other protein kinases described in the literature and suggest that activation by DNA is an important feature of the enzyme's in vivo function.

Abstract: Gap junction communication in some cells has been shown to be inhibited by pp60v-src, a protein tyrosine kinase encoded by the viral oncogene v-src. The gap junction protein connexin43 (Cx43) has been shown to be phosphorylated on serine in the absence of pp60v-src and on both serine and tyrosine in cells expressing pp60v-src. However, it is not known if the effect of v-src expression on communication results directly from tyrosine phosphorylation of the Cx43 or indirectly, for example, by activation of other second-messenger systems. In addition, the effect of v-src expression on communication based on other connexins has not been examined. We have used a functional expression system consisting of paired Xenopus oocytes to examine the effect of v-src expression on the regulation of communication by gap junctions comprised of different connexins. Expression of pp60v-src completely blocked the communication induced by Cx43 but had only a modest effect on communication induced by connexin32 (Cx32). Phosphoamino acid analysis showed that pp60v-src induced tyrosine phosphorylation of Cx43, but not Cx32. A mutation replacing tyrosine 265 of Cx43 with phenylalanine abolished both the inhibition of communication and the tyrosine phosphorylation induced by pp60v-src without affecting the ability of this protein to form gap junctions. These data show that the effect of pp60v-src on gap junctional communication is connexin specific and that the inhibition of Cx43-mediated junctional communication by pp60v-src requires tyrosine phosphorylation of Cx43.

Abstract: Protein phosphorylation is one of the most common mechanisms for controlling protein function. We now know that most phosphoproteins contain multiple phosphorylation sites and that these sites are often located in clusters. From the study of the enzyme glycogen synthase, one mechanism for the formation of phosphorylation clusters has been discovered that involves the concerted action of two or more protein kinases. One protein kinase, the primary kinase, introduces a phosphate group that is a requirement for the action of another, secondary, protein kinase. Thus the multiple phosphorylation occurs in a hierarchal fashion. This mechanism, which is critical for the phosphorylation of glycogen synthase, is likely to be a much more widespread phenomenon.

Abstract: Cyclic AMP-regulated gene expression is mediated by specific phosphoproteins (CREBs) which bind to cAMP-responsive elements of gene promoters. By analyzing the transactivation activities and phosphorylations in vivo of deletion and point mutated chimeric fusion proteins of the placental CREB-327, in which the DNA-binding domain is replaced by the heterologous binding-domain of the yeast transcription factor GAL4, we localized the cAMP-responsive and phosphorylated domain to a minimal-essential sequence module of 46 amino acids (residues 92-137). This serine-rich, multiply-phosphorylated sequence consists of at least three interdependent subdomains required for transcriptional activation. Although phosphorylation of serine-119 by cyclic AMP-dependent protein kinase A is necessary for transcriptional activation, such activation requires both a phosphorylated heptadecapeptide domain located ten residues amino terminal to the serine-119 and an eleven-residue domain carboxyl terminal to the serine-119. Deletion of these two domains does not impair phosphorylation of serine-119. Further, deletion of the carboxyl-terminal domain does not alter phosphorylation of the heptadecapeptide domain. We propose that akin to the phosphorylation-dependent activation of enzymes, the transcriptional transactivation functions of CREB-327 involve a phosphorylation-dependent allosteric conformational mechanism.

Abstract: Antigen-induced cross-linking of IgE bound to its receptors at the surface of basophils or mast cells initiates a number of biochemical events culminating in the release of histamine-containing granules. In the present study, we investigated the possible involvement of tyrosine phosphorylation in signaling by the high-affinity IgE receptor (Fc epsilon RI). Cross-linking of Fc epsilon RI in rat basophilic leukemia cells (RBL-2H3) led to the phosphorylation of several proteins on tyrosine, the most prominent having a mass of 72 kDa. Tyrosine phosphorylation was rapid, detectable 1 min after stimulation, and correlated with both the time course and antigen dose for histamine release. Reversal of Fc epsilon RI cross-linking prevented continuation of the degranulation process and resulted in rapid loss of tyrosine phosphorylation. The receptor-mediated tyrosine phosphorylation was still induced in the absence of calcium in the medium. Depletion of protein kinase C with phorbol 12-myristate 13-acetate did not dramatically affect the tyrosine phosphorylation signal or the release of histamine. In contrast, the calcium ionophore A23187 induced histamine release in the absence of a perceptible increase in protein tyrosine phosphorylation. Thus, tyrosine phosphorylation is an early signal following Fc epsilon RI aggregation, independent of the exocytotic process itself. Taken together, our findings functionally link protein phosphorylation on tyrosine residues to Fc epsilon RI-mediated signal transduction leading to histamine release.

Abstract: We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.

Abstract: We have shown that the transcriptional activity of the protooncogene jun (c-jun) promoter is repressed by a transcription factor, the cAMP response element-binding protein (CREB). This repression can be alleviated when CREB is phosphorylated by the catalytic subunit of protein kinase A. Repression cannot be alleviated by a mutant CREB deficient in the protein kinase A phosphorylation site (M1 CREB Ser-133----Ala), suggesting that phosphorylation of CREB at this site is essential for the relief of repression. Repression by CREB requires its binding to the c-jun promoter. In NIH 3T3 cells stably expressing CREB, c-jun is no longer induced by serum, but this repression can be relieved by treatment of the cells with forskolin, an agonist of the adenylate cyclase pathway. Thus, CREB has a dual function, that of a repressor in the absence of phosphorylation and an activator when phosphorylated by protein kinase A.

Abstract: T and B lymphocyte antigen receptors exhibit single transmembrane spanning regions and very short, three to five amino acid, C-terminal cytoplasmic tails. Ligation of these receptors leads, apparently through GTP binding protein activation, to rapid stimulation of a polyphosphoinositide specific phosphodiesterase (PPI-PDE). T lymphocyte antigen receptors (alpha beta) are coupled to PPI-PDE via a receptor associated complex of membrane proteins, designated CD3. Although an analogous transducer complex is presumed to exist in B cells, no such structure has been defined. We utilized in vitro [32P]phosphorylation to identify and characterize a membrane immunoglobulin (mIg) associated phosphoprotein complex which appears to represent a B cell analog of CD3. The phosphoprotein complex consists of three N-glycosylated polypeptides which occur as disulfide linked dimers, non-covalently associated with mIg. The complex associated with mIgM (pp32, pp34 and pp37 subunits) differs from that associated with mIgD (pp33, pp34 and pp37 subunits), and the isotype specific phosphoprotein (pp32 or pp33) appears to exist as a disulfide linked heterodimer with either pp34 or pp37. Aluminum fluoride stimulates phosphorylation of all of the subunits, and at least one of the proteins is phosphorylated on a tyrosine residue(s).

Abstract: The ABA-induced MA12 cDNA from maize, which encodes a set of highly phosphorylated embryo proteins, was used to isolate the corresponding genomic clone. This gene, called RAB-17 (responsive to ABA), encodes a basic, glycine-rich protein (mol. wt. 17,164) containing a cluster of 8 serine residues, seven of them contiguous. It is a homologue of the rice RAB-21 gene (Mundy J, Chua NH, EMBO J 7; 2279-2286, 1988). Phosphoamino acid analysis of the isolated protein indicates that only the serine residues are phosphorylated and a putative casein-type kinase phosphorylatable sequence was identified in the protein. The pattern of expression and in vivo phosphorylation of the RAB-17 protein was studied during maize embryo germination and in calli of both meristematic or embryonic origin. ABA treatment induced the synthesis of RAB-17 mRNA and protein in calli, however, the RAB-17 proteins were found to be highly phosphorylated only in embryos.