The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages

Lucifer Yellow CH (LY) is an excellent probe for fluid-phase pinocytosis. It accumulates within the macrophage vacuolar system, is not degraded, and is not toxic at concentrations of 6.0 mg/ml. Its uptake is inhibited at 0 degree C. Thioglycollate-elicited mouse peritoneal macrophages were found to exhibit curvilinear uptake kinetics of LY. Upon addition of LY to the medium, there was a brief period of very rapid cellular accumulation of the dye (1,400 ng of LY/mg protein per h at 1 mg/ml LY). This rate of accumulation most closely approximates the rate of fluid influx by pinocytosis. Within 60 min, the rate of LY accumulation slowed to a steady-state rate of 250 ng/mg protein per h which then continued for up to 18 h. Pulse-chase experiments revealed that the reduced rate of accumulation under steady-state conditions was due to efflux of LY. Only 20% of LY taken into the cells was retained; the remainder was released back into the medium. Efflux has two components, rapid and slow; each can be characterized kinetically as a first-order reaction. The kinetics are similar to those described by Besterman et al. (Besterman, J. M., J. A. Airhart, R. C. Woodworth, and R. B. Low, 1981, J. Cell Biol. 91:716-727) who interpret fluid-phase pinocytosis as involving at least two compartments, one small, rapidly turning over compartment and another apparently larger one which fills and empties slowly. To search for processes that control intracellular fluid traffic, we studied pinocytosis after treatment of macrophages with horseradish peroxidase (HRP) or with the tumor promoter phorbol myristate acetate (PMA). HRP, often used as a marker for fluid-phase pinocytosis, was observed to stimulate the rate of LY accumulation in macrophages. PMA caused an immediate four- to sevenfold increase in the rate of LY accumulation. Both HRP and PMA increased LY accumulation by stimulating influx and reducing the percentage of internalized fluid that is rapidly recycled. A greater proportion of endocytosed fluid passes into the slowly emptying compartment (presumed lysosomes). These experiments demonstrate that because of the considerable efflux by cells, measurement of marker accumulation inaccurately estimates the rate of fluid pinocytosis. Moreover, pinocytic flow of water and solutes through cytoplasm is subject to regulation at points beyond the formation of pinosomes.     

Src triggers circular ruffling and macropinocytosis at the apical surface of polarized MDCK cells

We addressed the role of Src on cortical actin dynamics and polarized endocytosis in MDCK cells harboring a thermosensitive v-src mutant. Shifting monolayers established at 40 degrees C (non-permissive temperature) to 34 degrees C (permissive temperature) rapidly reactivated v-Src kinase, but tight junctions and cell polarity resisted for >6 h. At this interval, activated v-src was recruited on apical vesicles, induced cortactin-associated apical circular ruffles productive of macropinosomes, thereby accelerating apical pinocytosis by approximately fivefold. Ruffling and macropinosome formation were selectively abrogated by inhibitors of actin polymerization, phosphoinositide 3-kinase, phospholipase C, and phospholipase D, which all returned apical pinocytosis to the level observed at 40 degrees C, underscoring the distinct control of apical micropinocytosis and macropinocytosis. Src promoted microtubule-dependent fusion of macropinosomes to the apical recycling endosome (ARE), causing its strong vacuolation. However, preservation of tubulation and apical polarity indicated that its function was not affected. The ARE was labeled for v-src, Rab11, and rabankyrin-5 but not early endosome antigen 1, thus distinguishing two separate Rab5-dependent apical pathways. The mechanisms of Src-induced apical ruffling and macropinocytosis could shed light on the triggered apical enteroinvasive pathogens entry and on the apical differentiation of osteoclasts.     

The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda

The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.     

Constitutive apical secretion of an 80-kD sulfated glycoprotein complex in the polarized epithelial Madin-Darby canine kidney cell line

The biosynthesis, processing, and apical secretion of a group of polypeptides (Kondor-Koch, C., R. Bravo, S. D. Fuller, D. Cutler, and H. Garoff. 1985. Cell. 43:297-306) are studied in MDCK cells using a specific polyclonal antiserum. These polypeptides are synthesized as a precursor protein which has an apparent Mr of 65,000 in its high mannose form. This precursor is converted into a protein with an apparent Mr of 80,000 containing complex carbohydrates and sulfate. After intracellular cleavage of the 80-kD protein, the 35-45-kD subunits are secreted as an 80-kD glycoprotein complex (gp 80) linked together by disulfide bonds. Secretion of the protein complex occurs by a constitutive pathway at the apical surface of the epithelial monolayer. Since the immediate post-translational precursor, the 65-kD protein, is hydrophilic in nature as shown by its partitioning behavior in a phase-separated Triton X-114 solution, gp 80 is segregated into the apical exocytotic pathway as a soluble molecule. The proteolytic maturation of gp 80 is blocked in the presence of chloroquine and its secretion is retarded. The 80-kD precursor is released at the apical cell surface, demonstrating that proteolytic processing is not necessary for the apical secretion of this protein. If N-glycosylation is inhibited by tunicamycin treatment the protein is secreted in equal amounts at both cell surfaces, indicating a role of the carbohydrate moieties in the vectorial transport of this protein.     

Schistocephalus solidus: pinocytosis by the plerocercoid tegument

The possibility of pinocytosis occurring in the tegument of the plerocercoid of Schistocephalus solidus has been investigated by morphological and experimental methods. Electronmicroscopic study showed that the outer syncytial tegument contained numerous electronlucid vesicles. These vesicles had two gradients, the number of vesicles decreasing from the outer canopy region to the inner canopy and from the apical to the basal plasma membrane for any particular region of tegument. A variety of morphological modifications of the apical plasma membrane very similar to those which have been accepted as evidence of pinocytosis in other tissues were present. In vitro studies using horseradish peroxidase, ruthenium red, and lanthanum nitrate showed that all three tracers are taken up by the tegument into membrane-limited vesicles. Vesicles which contained ruthenium red occurred at the base of the tegument within a 5-min incubation period, and their contents appeared to be released into the underlying interstitial material by exocytosis.     

Specific proteins in stem meristems during transition to flowering

Immunodiffusion tests were used for studying protein composition of apical buds ofRudbeckia bicolor andPerilla nankinensis during their transition from vegetative to reproductive state under inductive photoperiodic conditions or GA₃ treatment. In both species the induced buds differ from the vegetative ones in the presence of specific proteins (P): P1, P2, P3 appear inRudbeckia apical buds 2, 8, 16 d after the start of inductive treatment; P4 appears inPerilla apical buds 6 d after inductive treatment. P1, P2, P4 are revealed in induced buds in the early period of apex development when morphogenetic changes are not yet present. The similarity between antigenic spectra of induced buds and of those treated by GA₃ appears only inRudbeckia. These observations support the hypothesis of a change in gene expression at floral evocation. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

A transferrin-like GPI-linked iron-binding protein in detergent- insoluble noncaveolar microdomains at the apical surface of fetal intestinal epithelial cells

A GPI-anchored 80-kD protein was found to be the major component of detergent-insoluble complexes, prepared from fetal porcine small intestine, constituting about 25% of the total amount of protein. An antibody was raised to the 80-kD protein, and by immunogold electron microscopy of ultracryosections of mucosal tissue, the protein was localized to the apical surface of the enterocytes, whereas it was absent from the basolateral plasma membrane. Interestingly, it was mainly found in patches of flat or invaginated apical membrane domains rather than at the surface of microvilli. Caveolae were not found in association with these labeled microdomains. In addition, the 80-kD protein was seen in apical endocytic vacuoles and in tubulo-vesicular structures, suggesting that the apical microdomains are involved in endocytosis of the 80-kD protein. By its NH2-terminal amino acid sequence, iron-binding capacity and partial immunological cross- reactivity with serum transferrin, the 80-kD protein was shown to belong to the transferrin family, and it is probably homologous to melanotransferrin, a human melanoma-associated antigen. The 80-kD iron- binding protein was fully detergent-soluble immediately after synthesis and only became insoluble after gaining resistance to endo H, supporting a mechanism for exocytic delivery to the apical cell surface by way of detergent-insoluble glycolipid "rafts" that fuse with the plasmalemma at restricted sites devoid of microvilli.     

Rab11-FIP2 regulates differentiable steps in transcytosis

Transcytosis through the apical recycling system of polarized cells is regulated by Rab11a and a series of Rab11a-interacting proteins. We have identified a point mutant in Rab11 family interacting protein 2 (Rab11-FIP2) that alters the function of Rab11a-containing trafficking systems. Rab11-FIP2(S229A/R413G) or Rab11-FIP2(R413G) cause the formation of a tubular cisternal structure containing Rab11a and decrease the rate of polymeric IgA transcytosis. The R413G mutation does not alter Rab11-FIP interactions with any known binding partners. Overexpression of Rab11-FIP2(S229A/R413G) alters the localization of a subpopulation of the apical membrane protein GP135. In contrast, Rab11-FIP2(129-512) alters the localization of early endosome protein EEA1. The distributions of both Rab11-FIP2(S229A/R413G) and Rab11-FIP2(129-512) were not dependent on the integrity of the microtubule cytoskeleton. The results indicate that Rab11-FIP2 regulates trafficking at multiple points within the apical recycling system of polarized cells. Rab11a; immunoglobulin A; trafficking; apical recycling; GP135; early endosome; EEA1; Eps15 homology domain     

A photolabile oligodeoxyribonucleotide probe of the peptidyltransferase center: identification of neighboring ribosomal components

In this work we report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe and its exploitation in identifying 50S ribosomal subunit components neighboring its target site in 23S rRNA. The probe is complementary to 23S rRNA nucleotides 2497-2505, a single-stranded sequence that has been shown to fall within the peptidyltransferase center of Escherichia coli ribosomes [Cooperman, B. S., Weitzmann, C. J., & Fernandez, C. L. (1990) in The Ribosome: Structure, Function, & Evolution (Hill, W. E., Dahlberg, A., Garrett, R. A., Moore, P. B., Schlesinger, D., & Warner, J. R., Eds.) pp 491-501, American Society of Microbiology, Washington]. On photolysis in the presence of 50S ribosomes, it site-specifically incorporates into protein L3 (identified by both SDS-PAGE and immunological methods) and into three separate 23S rRNA regions: specifically, nucleotides 2454; 2501, 2502, 2505, 2506; and 2583, 2584. These results provide clear evidence that G-2505 in 23S rRNA is within 24 A (the distance between G-2505 and the photogenerated nitrene) of protein L3 and of each of the nucleotides mentioned above and are of obvious importance in the construction of detailed three-dimensional models of ribosomal structure. The approach we present is general and can be applied to determining ribosomal components neighboring regions of rRNA that are susceptible to binding by complementary oligodeoxyribonucleotides, both in intact 30S and 50S subunits and in subunits at various stages of reconstitution.     

Effect of different factors on the germination of spores in S and P- variants of Bacillus brevis]

The effect of temperature, duration of heating and the presence of L-alanine and L-glutamine in the medium on the spore germination was studied with the S and P- variants of Bacillus brevis which did not contain gramicidin S and with the R and P+ varants obtained on a defined medium with beta-phenyl-beta-alanine, an inhibitor of the biosynthesis of gramicidin S. The experiments were carried out according to the scheme of complete factor experiment. Germination of the spores was found upon their incubation in a defined medium with L-alanine within two hours after their preliminary heating at 80 degrees C during 45 minutes (S variant), at 60 degrees C during 45 minutes (R variant+trace amounts of gramicidin S), at 80 degrees C during 15 minutes (P+ variant/trace amounts of gramicidin S). Germination of the spores of the P- variant was best upon heating to 60 degrees C during 45 minutes. Gramicidin S is presumed to inhibit, to a certain extent, germination of the spores of its producing culture.     

Studies on the efficacy of a phytohormone producing phosphate solubilizingBacillus firmus in augmenting paddy yield in acid soils of Nagaland

Summary A super strain ofBacillus firmus (NCIM-2636) producing a phytohormone, indole-3-acetic acid, in addition to its high ability to solubilize insoluble inorganic phosphates were applied in acid soils of Nagaland, India. Rice (Oryza sativa L.) variety Jaya and IR-8 were grown in kharif season in two successive years 1980 and 1981. After proper manuring the soils received single super phosphate (S.S.P.) and Mussoorie Rock phosphate (R.P.) separately at different doses. Yield of crop in both the years increased significantly due to bacterial inoculation. Maximum grain yield was recorded in Jaya variety under S.S.P. and R.P. when treatments were at the dose of 43.75 and 17.5 kg P ha⁻¹ respectively while the same in IR-8 variety under S.S.P. and R.P. treatments were at the dose of 35 and 17.5 kg P ha⁻¹ respectively. Maximum straw yield was produced by Jaya variety when 35 and 43.75 kg P ha⁻¹ in the form of S.S.P. and R.P. respectively were applied. Highest straw yield of IR-8 variety was obtained after the application of 17.5 kg P ha⁻¹ (S.S.P. and R.P.) in combination with phosphate solubilizing bacteria. Bacterial inoculation decreased the phosphorus availability in 1 st year but increased the same in 2nd year. Phosphorus content in grains was significantly enhanced in both the trials. Maximum uptake of phosphorus by grains was noted in Jaya variety at the dose of 47.5 kg P ha⁻¹ and in IR-8 variety at the dose of 52.5 kg P ha⁻¹ under S.S.P. treatment, while 8.75 and 35 kg P ha⁻¹ in the form of R.P. yielded similar results in Jaya and IR-8 varieties respectively. Phosphorus at the dose of 35 kg ha⁻¹ was found to cause more P-uptake by straw in both S.S.P. and R.P. treatments. The various data from the experiment conclusively proved that the bacterium in combination with R.P. produced the desired effect more prominently than when bacterium applied in combination with S.S.P.     

Differential arrival of newly synthesized apical and basolateral plasma membrane proteins in the epithelial cell line A6

The labeling of specific cell surface proteins with biotin was used to examine both protein distribution and delivery of newly synthesized proteins to the apical and basolateral cell surface in A6 cells. Steady-state metabolic labeling with [³⁵S]methionine followed by specific cell surface biotinylation demonstrated polarization of membrane proteins. The delivery of newly synthesized proteins to the apical or basolateral cell surface was examined by metabolic labeling with [³⁵S]methionine using a pulse-chase protocol in combination with specific cell surface biotinylation. Newly synthesized biotinylated proteins at the apical cell surface reached a maximum after a 5 min chase, and then fell over the remainder of a 2 hr chase. The bulk flow of newly synthesized proteins to the basolateral membrane slowly rose to a maximum after 90 min. The detergent Triton X-114 was used to examine delivery of hydrophilic and hydrophobic proteins to the cell surface. Delivery of both hydrophilic and hydrophobic proteins to the apical cell surface reached a maximum 5 to 10 min into the chase period. The arrival of hydrophilic proteins at the basolateral surface showed early delivery and a maximum peak delivery at 120 min into the chase period. In contrast, only an early peak of delivery of newly synthesized hydrophobic proteins to the basolateral membrane was observed.This work was supported by grants from the American Heart Association, the National Kidney Foundation of the Delaware Valley, and from the Department of Veterans Affairs. T.R.K. is a recipient of an Established Investigatorship Award from the American Heart Association.     

Effects of a temperature sensitivity mutation in the J1R protein component of a complex required for vaccinia virus assembly

Vaccinia virus J1R protein is required for virion morphogenesis (W. L. Chiu and W. Chang, J. Virol. 76:9575-9587, 2002). In this work, we further characterized the J1R protein of wild-type vaccinia virus and compared it with the protein encoded by the temperature-sensitive mutant virus Cts45. The mutant Cts45 was found to contain a Pro-to-Ser substitution at residue 132 of the J1R open reading frame, which is responsible for a loss-of-function phenotype. The half-life of the J1R-P132S mutant protein was comparable at both 31 and 39 degrees C, indicating that the P132S mutation did not affect the stability of the J1R protein. We also showed that the J1R protein interacts with itself in the virus-infected cells. The N-terminal region of the J1R protein, amino acids (aa) 1 to 77, interacted with the C-terminal region, aa 84 to 153, and the P132 mutation did not abolish this interaction, as determined by two-hybrid analysis. Furthermore, we demonstrated that J1R protein is part of a viral complex containing the A30L, G7L, and F10L proteins in virus-infected cells. In immunofluorescence analyses, wild-type J1R protein colocalized with the A30L, G7L, and F10L proteins in virus-infected cells but the loss-of-function P132 mutant did not. Furthermore, without a functional J1R protein, rapid degradation of A30L and the 15-kDa forms of the G7L and F10L proteins was observed in cells infected with Cts45 at 39 degrees C. This study thus demonstrated the importance of the J1R protein in the formation of a viral assembly complex required for morphogenesis.     

Effects of energy or protein restriction followed by realimentation on the composition of gain and meat quality characteristics of Musculus longissimus dorsi in pigs

The effect of realimentation of fattening pigs on gain composition and intramuscular fat content in the longissimus dorsi muscle and their relationship to meat tenderness was investigated in 60 gilts. From day 90 to 168 of age, Group C (control) was fed according to the requirements, while from day 90 to 118 of age, Group R was fed restrictively (protein and energy) and Group P was fed restrictively with protein alone. This period was followed by adequate feeding. Comparative slaughter techniques were used; animals were slaughtered at the beginning of the study (day 90), at the end of the restriction period (day 118), and after two periods of realimentation (day 146 and day 168). Interactions between experimental treatments indicated that muscle protein gain (p < 0.05), muscle fat gain (p < 0.01), and shear force (p < 0.01) differed depending on period of realimentation. Muscle protein deposition differed only during the second period of realimentation, where Group R deposited more protein compared with Groups C and P (6.0, 4.5 and 5.0 g/d, respectively). Muscle fat deposition during the first period of realimentation was lower in Groups R and P than in Group C (0.73, 0.74 and 1.20 g/d, respectively) while in the second period Group R deposited considerably more fat than Groups C and P (2.39, 1.20 and 1.23 g/d, respectively). At the end of the first period, the shear force of Groups R and P was lower when compared with Group C (2.44, 2.79 and 3.22 kg, respectively); however, at final age it did not differ between treatments.     

Post-translational Modifications of Chicken Myelin Basic Protein Charge Components

Purified myelin basic protein (MBP) from various species contains several post-translationally modified forms termed charge components or charge isomers. Chicken MBP contains four charge components denoted as C1, C2, C3 and C8. (The C8 isomer is a complex mixture and was not investigated in this study.) These findings are in contrast to those found for human, bovine and other mammalian MBP’s. Mammalian MBP’s, each of which contain seven or eight charge components depending on the analysis of the CM-52 chromatographic curves and the PAGE gels obtained under basic pH conditions. Chicken MBP components C1, C2 and C3 were treated with trypsin and endoproteinase Glu-C. The resulting digests were analyzed by capillary liquid chromatography combined with either an ion trap tandem mass spectrometer or with a Fourier transform ion cyclotron resonance mass spectrometer. This instrumentation permitted establishing the amino acid composition and the determination of the post-translational modifications for each of the three charge components C1-C3. With the exception of N-terminal acetylation, the post-translational modifications were partial. The C1 component lacks any phosphorylated sites, a finding in agreement with the analysis of other MBP species. It also had a single methylation at R105 as did the components C2 and C3. The C2 component contains ten phosphorylated sites (S7, S18, S33, S64, S73, T96, S113, S141, S164, and S168), and modified arginine to citrulline residues at R24, and R165. Component C3 contains eight phosphorylated sites (S7, S33, S64, T96, S113, S141, S164, and S168), and citrulline residues at Arginine 41, R24 and R165. Partial deamidation of glutamine residues Q71, Q101 and Q146 were present in addition to asparagine N90 that was found in all three charge components. The glutamine at residue 3 is partially deamidated in isomers C1 and C2, whereas glutamine 74 and asparagine 83 were found not to be deamidated. Comparison of the PTM’s of MBP’s isolated from several vertebrate species reveals marked differences in their phosphate content. Chicken MBP does not share any phosphorylated sites with dogfish MBP; However, it does contain phosphorylated serine and threonine residues in common with mammalian MBP.     

Key Connexin 43 Phosphorylation Events Regulate the Gap Junction Life Cycle

Connexin 43 (Cx43), the most widely expressed and abundant vertebrate gap junction protein, is phosphorylated at multiple different serine residues during its life cycle. Cx43 is phosphorylated soon after synthesis and phosphorylation changes as it traffics through the endoplasmic reticulum and Golgi to the plasma membrane, ultimately forming a gap junction structure. The electrophoretic mobility of Cx43 changes as the protein proceeds through its life cycle, with prominent bands often labeled P0, P1 and P2. Many reports have indicated changes in “phosphorylation” based on these mobility shifts and others that occur in response to growth factors or other biological effectors. Here, we indicate how phosphospecific and epitope-specific antibodies can be utilized to show when and where certain phosphorylation events occur during the Cx43 life cycle. These reagents show that phosphorylation at S364 and/or S365 is involved in forming the P1 isoform, an event that apparently regulates trafficking to or within the plasma membrane. Phosphorylation at S325, S328 and/or S330 is necessary to form a P2 isoform; and this phosphorylation event is present only in gap junctions. Treatment with protein kinase C activators led to phosphorylation at S368, S279/S282 and S262 with a shift in mobility in CHO, but not MDCK, cells. The shift was dependent on mitogen-activated protein kinase activity but not phosphorylation at S279/S282. However, phosphorylation at S262 could explain the shift. By defining these phosphorylation events, we have begun to sort out the critical signaling pathways that regulate gap junction function.     

Phosphorylation of the amino-terminal head domain of the middle molecular mass 145-kDa subunit of neurofilaments. Evidence for regulation by second messenger-dependent protein kinases

To begin to understand the regulation and roles of neurofilament phosphorylation, we localized the phosphorylated domains on the 140-145-kDa neurofilament subunit (NF-M) and identified the protein kinases that may specifically phosphorylate the sites within these domains in vivo. Mouse retinal ganglion cells were labeled in vivo by injecting mice intravitreally with [32P]orthophosphate, and neurofilament-enriched fractions were obtained from the optic axons. Two-dimensional phosphopeptide map analysis of NF-M after digestion with alpha-chymotrypsin and trypsin revealed seven major (M8-M14) and at least eight minor (M1-M7 and M15) phosphopeptides. Two-dimensional phosphopeptide map analyses of NF-M phosphorylated in vitro by individual purified or endogenous axonal cytoskeleton-associated protein kinases showed that five peptides (M9-M13) were substrates for the heparin-sensitive second messenger-independent protein kinase(s). Protein kinase A and/or protein kinase C phosphorylated eight other peptides (M1-M8). Two alpha-chymotryptic peptides (C1 and C2) that were phosphorylated by protein kinase A but not by the endogenous independent kinase(s) were isolated by high performance liquid chromatography on a reverse-phase C8 column. Partial sequence analysis of peptides C1 (S R V S G P S ...) and C2 (S R G S P S T V S ...) showed that the peptides were localized on the head domain of NF-M at 25 and 41 residues from the amino terminus, respectively. Tryptic digest of peptide C1 (less than 12 kDa) generated the phosphopeptides M1-M6. Peptide C2 was a breakdown product of peptide C1. Since the polypeptide sites targeted by second messenger-independent kinase(s) associated with neurofilaments are localized on the carboxyl-terminal domain, separate aspects of NF-M function appear to be regulated by separate kinase systems that selectively phosphorylate head or tail domains of the polypeptide.