Phosphorylation of basic fibroblast growth factor by purified protein kinase C and the identification of a cryptic site of phosphorylation

We have further characterized the protein kinase C (PK-C) dependent phosphorylation of basic fibroblast growth factor (FGF). Intact recombinant basic FGF and a series of ten peptide fragments of basic FGF were phosphorylated by PK-C and the products were analyzed by SDS-PAGE and autoradiography. As expected, peptide fragments containing the known site of phosphorylation (Ser64) are substrates for phosphorylation. Surprisingly however, peptides containing the receptor binding domain of the mitogen [basic FGF(106-115)] are also phosphorylated. An examination of this sequence reveals the presence of a consensus sequence (Ser108-Ala109-Lys110) that mediates the reaction. Accordingly, all peptides that contain the core amino acids basic FGF(106-111) are substrates for phosphorylation. Peptide mapping of basic FGF confirms that Ser64 is the primary site of phosphorylation, suggesting that Ser108 is a cryptic consensus sequence. Because basic FGF is metabolized to sequence specific fragments after its binding and internalization into target cells, this cryptic site may in fact be phosphorylated in vivo.     

Regulation of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3

NDR protein kinases are involved in the regulation of cell cycle progression and morphology. NDR1/NDR2 protein kinase is activated by phosphorylation on the activation loop phosphorylation site Ser281/Ser282 and the hydrophobic motif phosphorylation site Thr444/Thr442. Autophosphorylation of NDR is responsible for phosphorylation on Ser281/Ser282, whereas Thr444/Thr442 is targeted by an upstream kinase. Here we show that MST3, a mammalian Ste20-like protein kinase, is able to phosphorylate NDR protein kinase at Thr444/Thr442. In vitro, MST3 selectively phosphorylated Thr442 of NDR2, resulting in a 10-fold stimulation of NDR activity. MOB1A (Mps one binder 1A) protein further increased the activity, leading to a fully active kinase. In vivo, Thr442 phosphorylation after okadaic acid stimulation was potently inhibited by MST3KR, a kinase-dead mutant of MST3. Knockdown of MST3 using short hairpin constructs abolished Thr442 hydrophobic motif phosphorylation of NDR in HEK293F cells. We conclude that activation of NDR is a multistep process involving phosphorylation of the hydrophobic motif site Thr444/2 by MST3, autophosphorylation of Ser281/2, and binding of MOB1A.     

Phosphorylation of HMG-I by protein kinase C attenuates its binding affinity to the promoter regions of protein kinase C gamma and neurogranin/RC3 genes

A 20-kDa DNA-binding protein that binds the AT-rich sequences within the promoters of the brain-specific protein kinase C (PKC) gamma and neurogranin/RC3 genes has been characterized as chromosomal nonhistone high-mobility-group protein (HMG)-I. This protein is a substrate of PKC alpha, beta, gamma, and delta but is poorly phosphorylated by PKC epsilon and zeta. Two major (Ser44 and Ser64) and four minor phosphorylation sites have been identified. The extents of phosphorylation of Ser44 and Ser64 were 1:1, whereas those of the four minor sites all together were <30% of the major one. These PKC phosphorylation sites are distinct from those phosphorylated by cdc2 kinase, which phosphorylates Thr53 and Thr78. Phosphorylation of HMG-I by PKC resulted in a reduction of DNA-binding affinity by 28-fold as compared with 12-fold caused by the phosphorylation with cdc2 kinase. HMG-I could be additively phosphorylated by cdc2 kinase and PKC, and the resulting doubly phosphorylated protein exhibited a >100-fold reduction in binding affinity. The two cdc2 kinase phosphorylation sites of HMG-I are adjacent to the N terminus of two of the three predicted DNA-binding domains. In comparison, one of the major PKC phosphorylation sites, Ser64, is adjacent to the C terminus of the second DNA-binding domain, whereas Ser44 is located within the spanning region between the first and second DNA-binding domains. The current results suggest that phosphorylation of the mammalian HMG-I by PKC alone or in combination with cdc2 kinase provides an effective mechanism for the regulation of HMG-I function.     

Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin by Cytoskeletal-Associated Intermediate Filament Protein Kinase Activity in Astrocytes

These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of basic fibroblast growth factor by a protein kinase associated with the outer surface of a target cell.

A protein kinase capable of phosphorylating basic fibroblast growth factor (FGF) can be localized on the outer cell surface of human hepatoma cells (SK-Hep cells). The addition of [gamma-32P]ATP, but not H3(32)PO4, results in a rapid (less than 10 min) incorporation of 32P into exogenously added basic FGF. The reaction is time and concentration dependent (apparent Km, 170 nM) and is stimulated by the addition of cAMP (EC50, 0.5 microM), but not the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate. There is also no tyrosine protein kinase detected on the cell surface. The inhibition of basic FGF binding to its low and/or high affinity sites decreases the phosphorylation of basic FGF by the ecto-protein kinase. Accordingly, pretreatment of cells with heparinase for 30 min or coincubation with heparin (0.1-10 micrograms/ml) decreases phosphorylation in a dose-dependent manner. Furthermore, the addition of a nonphosphorylatable peptide analog of basic FGF ([Val112] basic FGF-(106-146)NH2) that can compete with basic FGF binding to cells prevents the phosphorylation of basic FGF. Together, these observations suggest that 1) exogenous basic FGF must associate with its low and/or high affinity binding sites to be phosphorylated, and 2) the kinase is cAMP dependent and associated with the outer cell surface, and support the hypothesis that phosphorylation may regulate the activity and/or bioavailability of the growth factor.     

Phosphorylation of laminin-1 by protein kinase C

Several extracellular proteins have been reported to be phosphorylated. Previous studies of our laboratory indicated that laminin-1 can be phosphorylated by protein kinase A (PKA). Moreover, it has been reported that protein kinase C (PKC), although known to be intracellular, can phosphorylate extracellular proteins in the case of cellular damage and/or platelet activation. In the present study we examined the possibility of laminin-1 serving as a substrate of PKC. Amino acid analysis revealed that laminin-1 is phosphorylated by this enzyme on serine residues. Self assembly, heparin binding, and cell attachment on the phosphorylated molecule were then studied. Phosphorylated laminin-1 showed an increased and more rapid self assembly than the non-phosphorylated molecule. Heparin binding and cell attachment experiments indicated enhanced heparin and cell binding capacity of the phosphorylated molecule in comparison to the non- phosphorylated control. These results indicate that laminin-1 can be phosphorylated by protein kinase C. Furthermore, phosphorylation by protein kinase C seems to alter several properties of the molecule, though, the in vivo significance of this phenomenon remains to be studied.     

An analysis of the substrate specificity of insulin-stimulated protein kinase-1, a mammalian homologue of S6 kinase-II

The specificity determinants for insulin-stimulated protein kinase-I (ISPK-1) have been investigated with synthetic peptides based on naturally-occurring protein phosphoacceptor sequences. Peptides (Arg-Arg-Xaa-Ser-Xaa) that fulfill the consensus sequence for cyclic-AMP-dependent protein kinase (PK-A) are also phosphorylated readily by ISPK-1. The phosphorylation efficiency is improved by increasing the number of N-terminal arginine residues and by moving the arginyl cluster one residue further away from the serine, the nonapeptide (Arg)₄-Ala-Ala-Ser-Val-Ala being the best substrate among all the short peptides tested (K_m = 15 μM). Conversely, the substitution of either Thr for Ser or Lys for Arg is detrimental. Likewise, two flanking Pro residues and an Arg immediately N-terminal to the Ser act as negative specificity determinants. While the specificity of ISPK-1 shows several similarities to that of PK-A, including an absolute requirement for basic residues on the N-terminal side of the target Ser, it differs in several other respects including (1), the detrimental effect of a Lys for Arg substitution which is still compatible with some phosphorylation by ISPK-1, but not PK-A; (2), the presence of C-terminal acidic residues which are tolerated very well by ISPK-1, but are detrimental to PK-A; (3), the effect of substituting Phe for Val in the peptide Arg-Arg-Ala-Ser-Val-Ala, which improves the efficiency of phosphorylation by PK-A (lowering the K_m 4-fold), but has no effect on phosphorylation by ISPK-1. These differences in peptide substrate specificity may account in part for the different rates of phosphorylation of physiological substrates for ISPK-1 and PK-A, such as the G subunit of protein phosphatase-1.     

Effects of protein kinase inhibitors on pig oocyte maturation in vitro.

Normal oocyte maturation depends on signal transmission between granulosa cells and the oocyte. We have analysed the effects of inhibiting (I) cyclic AMP-dependent protein kinase (protein kinase A, PK-A), (II) Ca2+/phospholipid-dependent protein kinase (protein kinase C, PK-C) and (III) calmodulin (CaM) on pig oocyte maturation in vitro, protein synthesis and phosphorylation. The inhibition of PK-A using a specific inhibitor H8, decreased the maturation rate (rate of germinal vesicle breakdown, GVBD) of cumulus-enclosed pig oocytes in a dose-dependent manner by approximately 12%, reaching a plateau at 100 microM. The inhibition of PK-C with H7, an inhibitor with some side-effects on PK-A, decreased the maturation rate of cumulus-enclosed oocytes in a dose-dependent manner to a maximum of 20% at a concentration of 100 microM. The calmodulin antagonist W7 up to a concentration of 200 microM had no effects on maturation of cumulus-enclosed pig oocytes. None of the inhibitors (H7, H8 and W7) altered the patterns of protein synthesis of either pig oocytes and cumulus cells after maturation in vitro. Oocyte phosphoprotein patterns were, however, clearly changed by W7. Cumulus cell protein phosphorylation patterns were changed by all 3 agents. Since inhibition of cyclic AMP and Ca2+ phospholipid pathways by PK-A and PK-C blocking chemicals affected only a limited proportion of oocytes (12 and 20%, respectively) and inhibition of Ca2+ binding to CaM was without effect on oocyte maturation, we conclude that these pathways modulate rather than regulate oocyte maturation in the pig.     

pp54 microtubule-associated protein 2 kinase. A novel serine/threonine protein kinase regulated by phosphorylation and stimulated by poly-L-lysine

An hepatic protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) on Ser/Thr residues is markedly activated after intraperitoneal injection of cycloheximide in the rat. The enzyme has been purified greater than 10,000-fold to near homogeneity and corresponds to a 54-kDa polypeptide, based on auto-phosphorylation, renaturation of activity from sodium dodecyl sulfate gels, and gel filtration. The protein kinase activity is unaffected by prior autophosphorylation, Ca2+, diacylglycerol and phospholipids, cyclic nucleotides, staurosporine, and protein kinase inhibitor, but can be totally and specifically deactivated by the Ser/Thr protein phosphatase 2A. The enzyme is inhibited completely but reversible by transition metals and p-chloromercuribenzoate, and is strongly stimulated by poly-L-lysine toward most, but not all protein substrates. The activity of the cycloheximide-stimulated MAP-2 kinase (pp54 MAP-2 kinase) toward potential polypeptide substrates was compared to that of an insulin-stimulated MAP-2 kinase (pp42 MAP-2 kinase). Although both MAP-2 kinases exhibited little or no ability to phosphorylate histones and casein, the two kinases had a distinguishable substrate specificity. At comparable MAP-2 phosphorylating activities, pp42 MAP-2 kinase, but not pp54 MAP-2 kinase, phosphorylated and activated the Xenopus S6 protein kinase II. Moreover, pp42 MAP-2 kinase phosphorylated myelin basic protein at 10-12-fold higher rates than did pp54 MAP-2 kinase. Cycloheximide-activated pp54 MAP-2 protein kinase appears to be a previously uncharacterized protein kinase that is itself regulated through Ser/Thr phosphorylation and, perhaps, polypeptide regulators with basic domains. The identity of the upstream regulatory elements and the native substrates remain to be established.     

Akt/PKB kinase phosphorylates separately Thr212 and Ser214 of tau protein in vitro

Microtubule-associated protein tau contains a consensus motif for protein kinase B/Akt (Akt), which plays an essential role in anti-apoptotic signaling. The motif encompasses the AT100 double phospho-epitope (Thr212/Ser214), a specific marker for Alzheimer's disease (AD) and other neurodegenerations, raising the possibility that it could be generated by Akt. We studied Akt-dependent phosphorylation of tau protein in vitro. We found that Akt phosphorylated both Thr212 and Ser214 in the longest and shortest tau isoforms as determined using phospho site-specific antibodies against tau. Akt did not phosphorylate other tau epitopes, including Tau-1, AT8, AT180, 12E8 and PHF-1. The Akt-phosphorylated tau retained its initial electrophoretic mobility. Immunoprecipitation studies with phospho-specific Thr212 and Ser214 antibodies revealed that only one of the two sites is phosphorylated per single tau molecule, resulting in tau immunonegative for AT100. Mixed kinase studies showed that prior Ser214 phosphorylation by Akt blocked protein kinase A but not GSK3beta activity. On the other hand, GSK3beta selectively blocked Ser214 phosphorylation, which was prevented by lithium. The results suggest that Akt may be involved in AD-specific phosphorylation of tau at the AT100 epitope in conjunction with other kinases. Our data suggest that phosphorylation of tau by Akt may play specific role(s) in Akt-mediated anti-apoptotic signaling, particularly relevant to AD and other neurodegenerations.     

Regulation of phosphorylation of tau by cyclin-dependent kinase 5 and glycogen synthase kinase-3 at substrate level

Microtubule associated protein tau, which is expressed in six alternatively spliced molecular isoforms in human brain, is abnormally hyperphosphorylated in Alzheimer disease and related tauopathies. Here, we show (i) that GSK-3alpha and neither GSK-3beta nor cdk5 can phosphorylate tau at Ser262 and phosphorylation at Ser235 by cdk5 primes phosphorylation at Thr231 by GSK-3alpha/beta; (ii) that tau isoforms with two N-terminal inserts (tau4L, tau3L) are phosphorylated by cdk5 plus GSK-3 at Thr231 markedly more than isoforms lacking these inserts (tau4, tau3); and (iii) that Thr231 is phosphorylated approximately 50% more in free tau than in microtubule-bound tau, and the phosphorylation at this site results in the dissociation of tau from microtubules. These findings suggest that the phosphorylation of tau at Thr231 and Ser262 by cdk5 plus GSK-3, which inhibits its normal biological activity, is regulated both by its amino terminal inserts and its physical state.     

Rum1, an inhibitor of cyclin-dependent kinase in fission yeast, is negatively regulated by mitogen-activated protein kinase-mediated phosphorylation at Ser and Thr residues.

The p25(rum1) is an inhibitor of Cdc2 kinase expressed in fission yeast and plays an important role in cell-cycle control. As its amino-acid sequence suggests that p25(rum1) has putative phosphorylation sites for mitogen-activated protein kinase (MAPK), we investigated the ability of MAPK to phosphorylate p25(rum1). Direct in vitro kinase assay using GST-fusion proteins of wild-type as well as various mutants of p25(rum1) demonstrated that MAPK phosphorylates the N-terminal portion of p25(rum1) and residues Thr13 and Ser19 are major phosphorylation sites for MAPK. In addition, phosphorylation of p25(rum1) by MAPK revealed markedly reduced Cdc2 kinase inhibitor ability of the protein. Together with the fact that replacement of both Thr13 and Ser19 with Glu, which mimics the phosphorylated state of these residues, also significantly reduces the activity of p25(rum1) as a Cdc2 inhibitor, it was suggested that the phosphorylation of Thr13 and Ser19 negatively regulates the function of p25(rum1). Further evidence indicates that phosphorylation of Thr13 and Ser19 may retain a negative effect on the function of p25(rum1) even in vivo. Therefore, MAPK may regulate the function of p25(rum1) via phosphorylation of its Thr and Ser residues and thus participate in cell cycle control in fission yeast.     

Importance of autophosphorylation at Ser186 in the A-loop of salt inducible kinase 1 for its sustained kinase activity

Autophosphorylation is an important mechanism by which protein kinases regulate their own biological activities. Salt inducible kinase 1 (SIK1) is a regulator in the feedback cascades of cAMP-mediated gene expression, while its kinase domain also features autophosphorylation activity. We provide evidence that Ser186 in the activation loop is the site of autophosphorylation and essential for the kinase activity. Ser186 is located at the +4 position of the critical Thr residue Thr182, which is phosphorylated by upstream kinases such as LKB1. The relationship between phosphorylation at Ser186 and at Thr182 in COS-7 cells indicates that the former is a prerequisite for the latter. Glycogen synthase kinase-3beta (GSK-3beta) phosphorylates Ser/Thr residues located at the fourth position ahead of the pre-phosphorylated Ser/Thr residues, and inhibitors of GSK-3beta reduce the phosphorylation at Thr182. The results of an in vitro reconstitution assay also indicate that GSK-3beta could be the SIK1 kinase. However, overexpression and knockdown of GSK-3beta in LKB1-defective HeLa cells suggests that GSK-3beta alone may not be able to phosphorylate or activate SIK1, indicating that LKB1 may play a crucial role by phosphorylating SIK1 at Thr182, possibly as an initiator of the autophosphorylation cascade, and GSK-3beta may phosphorylate SIK1 at Thr182 by recognizing the priming-autophosphorylation at Ser186 in cultured cells. This may also be the case for the other isoform SIK2, but not for SIK3.     

Characterization of dual specificity protein kinase from maize seedlings

A protein kinase of 57 kDa, able to phosphorylate tyrosine in synthetic substrates pol(Glu4,Tyr1) and a fragment of Src tyrosine kinase, was isolated and partly purified from maize seedlings (Zea mays). The protein kinase was able to phosphorylate exogenous proteins: enolase, caseins, histones and myelin basic protein. Amino acid analysis of phosphorylated casein and enolase, as well as of phosphorylated endogenous proteins, showed that both Tyr and Ser residues were phosphorylated. Phosphotyrosine was also immunodetected in the 57 kDa protein fraction. In the protein fraction there are present 57 kDa protein kinase and enolase. This co-purification suggests that enolase can be an endogenous substrate of the kinase. The two proteins could be resolved by two-dimensional electrophoresis. Specific inhibitors of typical protein-tyrosine kinases had essentially no effect on the activity of the maize enzyme. Staurosporine, a nonspecific inhibitor of protein kinases, effectively inhibited the 57 kDa protein kinase. Also, poly L-lysine and heparin inhibited tyrosine phosphorylation by 57 kDa maize protein kinase. The substrate and inhibitor specificities of the 57 kDa maize protein kinase phosphorylating tyrosine indicate that it is a novel plant dual-specificity protein kinase.     

p90(RSK) blocks bad-mediated cell death via a protein kinase C-dependent pathway

Although activation of protein kinase C (PKC) is known to promote cell survival and protect against cell death, the PKC targets and pathways that serve this function have remained elusive. Here we demonstrate that two potent activators of PKC, 12-O-tetradecanoylphorbol-13-acetate and bryostatin, both stimulate phosphorylation of Bad at Ser(112), a site known to regulate apoptotic cell death by interleukin-3. PKC inhibitors but not PI 3-kinase/Akt inhibitors block 12-O-tetradecanoylphorbol-13-acetate-stimulated Bad phosphorylation. PKC isoforms tested in vitro were unable to phosphorylate Bad at Ser(112), suggesting that PKC acts indirectly to activate a downstream Bad kinase. p90(RSK) and family members RSK-2 and RSK-3 are activated by phorbol ester and phosphorylate Bad at Ser(112) both in vitro and in vivo. p90(RSK) stimulates binding of Bad to 14-3-3 and blocks Bad-mediated cell death in a Ser(112)-dependent manner. These findings suggest that p90(RSK) can function in a PKC-dependent pathway to promote cell survival via phosphorylation and inactivation of Bad-mediated cell death.     

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.     

Identification of sites of phosphorylation by G-protein-coupled receptor kinase 2 in beta-tubulin.

G-protein-coupled receptor kinase 2 (GRK2) is known to specifically phosphorylate the agonist-bound forms of G-protein-coupled receptors (GPCRs). This strict specificity is due at least partly to activation of GRK2 by agonist-bound GPCRs, in which basic residues in intracellular regions adjacent to transmembrane segments are thought to be involved. Tubulin was found to be phosphorylated by GRK2, but it remains unknown if tubulin can also serve as both a substrate and an activator for GRK2. Purified tubulin, phosphorylated by GRK2, was subjected to biochemical analysis, and the phosphorylation sites in beta-tubulin were determined to be Thr409 and Ser420. In addition, the Ser444 in beta III-tubulin was also indicated to be phosphorylated by GRK2. The phosphorylation sites in tubulin for GRK2 reside in the C-terminal domain of beta-tubulin, which is on the outer surface of microtubules. Pretreatment of tubulin with protein phosphatase type-2A (PP2A) resulted in a twofold increase in the phosphorylation of tubulin by GRK2. These results suggest that tubulin is phosphorylated in situ probably by GRK2 and that the phosphorylation may affect the interaction of microtubules with microtubule-associated proteins. A GST fusion protein of a C-terminal region of beta I-tubulin (393-445 residues), containing 19 acidic residues but only one basic residue, was found to be a good substrate for GRK2, like full-length beta-tubulin. These results, together with the finding that GRK2 may phosphorylate synuclein and phosducin in their acidic domains, indicate that some proteins with very acidic regions but without basic activation domains could serve as substrates for GRK2.     

Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium.

A protein of apparent Mr = 15,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is the major plasma membrane substrate for cAMP-dependent protein kinase (PK-A) and protein kinase C (PK-C) in several different tissues. In the work described here, we purified, cloned, and sequenced the canine cardiac sarcolemmal "15-kDa protein." The amino terminus of the purified protein was not blocked, allowing determination of 50 consecutive residues by standard Edman degradation. Overlapping proteolytic phosphopeptides yielded 22 additional residues at the carboxyl terminus. Dideoxy sequencing of the full-length cDNA confirmed that the 15-kDa protein contains 72 amino acids, plus a 20-residue signal sequence. The mature protein has a calculated Mr = 8409. There is one hydrophobic membrane-spanning segment composed of residues 18-37. The acidic amino-terminal end (residues 1-17) of the protein is oriented extracellularly, whereas the basic carboxyl-terminal end (residues 38-72) projects into the cytoplasm. The positively charged carboxyl terminus contains the phosphorylation sites for PK-A and PK-C. In the transmembrane region, the 15-kDa protein exhibits 52% amino acid identity with the "gamma" subunit of Na,K-ATPase. High stringency Northern blot analysis revealed that 15-kDa mRNA is present in heart, skeletal muscle, smooth muscle, and liver but absent from brain and kidney. We propose the name "phospholemman" for the 15-kDa protein, which denotes the protein's location within the plasma membrane and its characteristic multisite phosphorylation.     

Characterization of the in vitro phosphorylation of human tau by tau protein kinase II (cdk5/p20) using mass spectrometry

Hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies, and tau protein kinase II is reported to play a role in the pathogenesis of Alzheimer's disease. Recently, we reported that tau protein kinase II (cdk5/p20)-phosphorylated human tau inhibits microtubule assembly, and tau protein kinase II (cdk5/p20) phosphorylation of microtubule-associated tau results in dissociation of phosphorylated tau from the microtubules and tubulin depolymerization. In the studies reported here, a combination of mass spectrometric techniques was used to study the phosphorylation of human recombinant tau by recombinant tau protein kinase II (cdk5/p20) in vitro. The extent of phosphorylation was determined by measuring the molecular mass of phosphorylated tau using mass spectrometry. Reaction of human recombinant tau with tau protein kinase II (cdk5/p20) resulted in the formation of two major species containing either five or six phosphate groups. The specific amino acid residues phosphorylated were determined by analyzing tryptic peptides by tandem mass spectrometry via either MALDI/TOF post-source decay or by electrospray tandem mass spectrometry. Based on these experiments, we conclude that tau protein kinase II (cdk5/p20) can phosphorylate human tau at Thr(181), Thr(205), Thr(212), Thr(217), Ser(396) and Ser(404).