Protein Phosphorylation in Mitochondria from

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This manuscript was published as, Corso, M., Thomson, M. (2001). Protein Phosphorylation in Mitochondria from Human Placenta. Placenta, 22, 432-439, and is available from http://www.placentajournal.org/

Protein Phosphorylation in Mitochondria from Human Placenta Monica Corso1 and Murray Thomson2

1. School of Science, University of Western Sydney Nepean, PO Box 10, Kingswood, NSW, 2747, Australia. 2. Corresponding author, School of Biological Sciences, University of Sydney, NSW, 2006, Australia. Phone +61 2 9351 5284, FAX +61 2 9351 2175, e-mail [email protected]

Abstract The aim of this study was to investigate whether mitochondria from human placenta contain phosphorylated proteins and kinases. Interestingly, the placenta contains two types of mitochondria with different sizes. These are ‘heavy’ mitochondria which sediment at a much lower g force than ‘light’ mitochondria. Mitochondria were incubated with [γ32]P-ATP and labelled proteins analysed by electrophoresis and autoradiography. A major protein band of 20 kDa was detected with minor bands at 22, 38 and 85 kDa. The 20 kDa band was attenuated by 83 % by the co-incubation of mitochondria with Herbimycin a tyrosine kinase inhibitor. A 20 kDa protein was also identified using an anti-tyrosine phosphate antibody and detection of this protein was significantly higher in heavy mitochondria as opposed to light mitochondria. Protein kinase A enzyme activity was also detected in mitochondria at a level not significantly different than that found in whole non fractionated cells.

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These data indicate that mitochondria from human placenta contains kinase activity and phosphoproteins. These molecules may have functions in signalling systems in this organelle. Phosphoprotein signalling systems may be differentially modulated in heavy mitochondria as compared to light mitochondria.

Introduction Reversible protein phosphorylation is used by signalling systems in many parts of the cell to activate proteins such as enzymes, cytoskeletal elements, ion channels, and nucleic acid regulatory factors. When activated by phosphorylation, effector proteins have been shown to exert profound effects on cell regions such as the plasma membrane, endoplasmic reticulum and the nucleus. By regulating events in different regions of the cell, phosphoproteins can modulate cellular metabolism, growth and differentiation (Greengard, 1976).

Protein phosphorylation is often catalyzed by one of the many protein kinase species. One of the earliest discovered of these is the cAMP-dependent kinase (PKA) (Brostron et al., 1970) which can migrate to many areas of the cell (Meinkoth 1990). PKA anchoring proteins (AKAPs) serve to keep the PKA holoenzyme in defined areas of the cell (Pawson and Scott, 1997).

In 1977 Takai and colleagues discovered Protein kinase C, a serine-threonine kinase with calcium and phosholipid dependant actions (Takai et al., 1977). PKC and can be moored in a defined cellular location by proteins including, receptors for activated C kinase (RACKS) (Csukai et al., 1999) and substrates that interact with C-kinase (STICKs) (Chapline et al., 1999). Inactive PKC can also bind to AKAPs (Klauk et al., 1996).

The mitogen-activated protein kinases (MAPK) form another group of serinethreonine kinases which phosphorylate transcription factors and stimulate growth by altering the expression of specific genes (Hardy and Chaudhri, 1997). MAPKs often function as part of a phosphorylation cascade and can be phosphorylated and 2

activated by MAPK kinases. The majority of MAPK kinases are located in the cytoplasm and phosphorylation of a MAPK in the cytoplasm allows the enzyme to travel to the nucleus where it can interact with a substrate (Lenormand et al., 1993).

As well as containing sites that can be phosphorylated by serine-threonine kinases, MAPKs can be targets for tyrosine kinases that have important roles in transmitting mitogenic signals (Mano, 1999). In addition to membrane spanning tyrosine kinases there are tyrosine kinases which do not have membrane anchor regions and these exist in aqueous portions of the cell. The name “non-receptor” tyrosine kinase is probably more appropriate than the term “cytosolic” tyrosine kinases to describe enzymes of this class as non-receptor tyrosine kinases can translocate to the nucleus (Neet and Hunter, 1996).

A number of recent studies have investigated the cellular locations of kinases in placental cells. PKC has shown to be localised in the plasma membrane (Ruzycky et al., 1996) of placental tissue and is believed to be involved in the control of human chorionic gonadotropin release (Baker et al., 1998). The cellular distribution of the regulatory subunit of PKA, RIα has been shown to be concentrated in the golgicentrosome area in cytotrophoblast whereas in the syncytiotrophoblast it is scattered throughout the cytoplasm (Yura et al., 1998).

Surprisingly, there is relatively little in the scientific literature on the role of protein phosphorylation systems in the mitochondrion. Nonetheless, several groups have begun investigating the possibility that protein phosphorylation may be exerted in the mitochondrion.

In isolated rat hepatocytes, glucagon has been shown to

promote phosphorylation of a 35 kDa mitochondrial protein (Vargas et al., 1982). In bovine heart mitochondria it has been demonstrated that a mitochondrial protein kinase A (PKA) is responsible for the phosphorylation of an 18 kDa protein (Papa et al., 1996).

Placental tissue would appear likely to contain phospho-protein signalling systems in the mitochondria as the placenta is an ephemeral organ which displays some unique 3

patterns of cell differentiation and cellular signalling. The growth and development of the placenta depends on the differentiation of a portion of trophoblast cells into syncytiotrophoblasts. It is only the syncytiotrophoblast cell that expresses the enzymes needed for producing steroid hormones that are essential for the foetus to be carried to term (Albrecht and Pepe, 1990). Interestingly, the mitochondria of the syncytiotrophoblast display a different morphology to that of mitochondria in the cytotrophoblast cells and syncytiotrophoblast mitochondria are lighter than those in cytotrophoblast (Martinez, 1996). In this project we investigated the possibility that mitochondria of the human placenta contain phosphorylating enzymes and substrates.

Materials and Methods

Mitochondrial Isolation from Human Placenta Human term placentae were collected from Westmead Hospital, NSW, Australia, immediately after delivery and placed in chilled phosphate buffered saline (PBS). Pieces of tissue approximately 1cm3 were cut from the mother’s side of the placenta and homogenized in buffer A containing; Tris-HCl 20 mM, mannitol 210 mM, and sucrose 70 mM. This fraction was referred to as whole placental extract. The homogenate was then centrifuged at 700 x g for 10 minutes at 4o C to remove cell debris and nuclei. The supernatant was decanted and kept at 4o C, the pellet was reextracted twice by re-suspension in buffer A and centrifuged again at 700 x g for 10 minutes at 4o C. Supernatants were then combined and centrifuged at 9750 x g for 15 minutes at 4o C to yield the mitochondrial pellet. Mitochondria were washed 3 times by re-suspension in approximately 200 ml of buffer A and centrifuged at 9750 x g at 4o C for 15 min. After the 3 washes the supernatants (fractions containing cytosol, plasma membrane and microsomes) were removed and the mitochondrial pellets resuspended in buffer A. This preparation is referred to simply as ‘mitochondria’. This method has been shown previously to yield mitochondria which are free from contaminants from other parts of the cell (Thomson, 1998a, Thomson

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et al., 1998). The mitochondria used in this study were checked for purity using antibodies to ras as previously described (Thomson, 1998, Thomson et al., 1998). Briefly, samples containing 50 µg of protein were analysed by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Western blot (see below) using an antibody to the protein ras (Santa Cruz). While antibodies to the ras protein did not show any binding in the 21 kDa range to any of the mitochondria purified by the method above, they revealed the presence of ras in all fractions containing cytosol, plasma membrane and microsomes (results not shown). This demonstrated that the mitochondria were not contaminated by fractions containing cytosol, plasma membrane and microsomes. In some experiments the mitochondrial fraction was separated into heavy and light mitochondria as described below.

Separation of Heavy and Light Mitochondria Isolated mitochondria from human placentae were separated by sequential centrifugation following the method of Martinez et al. (1996). Mitochondria were resuspended in an equal volume of buffer A and centrifuged at 4000 x g for 15 minutes at 4o C. The pellet was designated ‘heavy’ mitochondria. The supernatant was removed and further centrifuged at 16,000 x g for 15 minutes at 4o C to obtain ‘light’ mitochondria. Mitochondria that had been further purified by this process are referred to as light mitochondria and heavy mitochondria to differentiate these fractions from mitochondria that were not separated into light and heavy fractions.

Assay of Protein Concentration The protein concentration of mitochondrial samples from each placenta was determined using the Pierce BCA Protein micro assay and a Bio-Rad Model 550 microplate reader as per manufacturer instructions. Incubation of Mitochondria with [γ 32P]-ATP Phosphorylation of mitochondrial proteins was assayed using a modification of the method of Backer et al. (1986) as follows. Mitochondrial proteins were prepared in buffer B (20 mM Tris-HCl, 210 mM Mannitol, 70mM Sucrose, and 1 mM MgCl2) at a concentration of 200 µg/ml and 100 µl samples placed in the wells of a 96 well plate. To 5

each well 0.5 µCi [γ32P]-ATP (>5000 Ci/mmol, Sigma) was added unless stated otherwise. Reactions were terminated after 1 min unless stated otherwise by the addition of two volumes of SDS sample buffer.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE) Gel electrophoresis was performed using 12 % separating gels and the Biorad Mini Protean II system as per the manufacturer’s instructions. Mitochondrial and homogenised whole placental samples were first diluted in SDS sample buffer (0.5 M Tris-HCl pH 6.8, 10% SDS w/v, 10% glycerol, 5% 2-mercaptoethanol, and 1% bromophenol blue w/v) and then heated at 95o C for 5 minutes. Radioactive proteins separated by SDS PAGE were detected by autoradiography utilizing Kodak X-Omat film. Bands detected by autoradiography were analysed using the computer program NIH image.

Western Blot Proteins separated by SDS PAGE were transferred electrophoretically to a nitrocellulose membrane (1 hr at 100 volts) using a Mini Trans-Blot system (Bio-Rad laboratories) according to manufacturer’s instructions. The nitrocellulose membrane was then blocked with 5% skim milk in Tween in Tris buffered saline (TTBS; 0.05% Tween, 20 mM Tris and 0.9% NaCl, pH 7.5) for 1 hr. Following the blocking of nonspecific binding the nitrocellulose membrane was then incubated with a specific antibody to phosphotyrosine (4G10, Upstate Biotechnology) or ras (Santa Cruz) diluted 1/1000 with 5% skim milk in TTBS for 1 hr. After incubation with the primary antibody the membrane was washed 3 times for 5 minutes each with TTBS, and incubated with secondary antibody (anti-mouse horse radish peroxidase conjugate, Santa Cruz) diluted 1/3000 with 5% skim milk in TTBS for 45 minutes at room temperature. Following incubation with secondary antibody the membrane was washed 3 times for 5 minutes each with TTBS, and once for 5 minutes with Tris buffered saline (TBS). Western blot chemiluminescence was performed using the Western Blot Chemiluminescence Reagent Plus kit (NEN Life Science products), as per manufacturer’s instructions and exposed to X-Ray film.

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Protein Kinase A and C Assays PKA and PKC activity was measured using the Mesacup protein kinase assay system (Upstate Biotechnology) as per the manufacturer’s instructions. Samples containing 10 µg of protein in 12 µl of buffer A were analysed in the assay.

Immunoprecipitation Lysis buffer (0.4 ml) consisting of 20 mM TRIS-HCl ph 7.4. 5 mM EDTA, 137 mM NaCl, 1 mM MgCl2, 1 mM sodium vanadate, 1 mM PMSF, 1% nonidet P-40 was added to 100 µl mitochondria (at a protein concentration of 200µg /ml) incubated with [γ32P]– ATP as described above with the exception that SDS buffer was not added, to allow immune complexes to form. Antibodies to phosphotyrosine (2 µg, 4G10, Upstate Biotechnology) were then added and the mixture was incubated at 4o C for 60 minutes. The antibody complexes were incubated with 50 µl of 30% protein-ASepharose (Sigma) for 30 min at 4o C and immunoprecipitates obtained by centrifugation at 2000g for 10 minutes. The precipitate was then washed five times in chilled lysis buffer and analysed by SDS PAGE and autoradiography.

Statistical analysis Results were analysed by analysis of variance (ANOVA) and a p value of 0.05 or less was considered significant.

Results

Phosphorylation of mitochondrial proteins When mitochondria were exposed to [γ32P]-ATP and analysed by SDS PAGE, Western blot and autoradiography with 4 hours film exposure, a 20 kDa protein bands was identified (Figure 1). Very faint bands at 38 and 85 kDa could also be seen. A minor band at 22 kDa was also observed in some experiments but was not observed reproducibly in all experiments.

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Figure 2 shows the dose responsiveness of the labelling of the 20 kDa protein following incubation of mitochondria with 0.5, 0.2, 0.1, 0.06, 0.005 and 0.0005 µCi of [γ32P]-ATP.

The effects of protein kinase inhibitors on protein phosphorylation Mitochondria were incubated with [γ-32P]-ATP and with and without the following kinase inhibitors: chelerythrine (10 µM) a PKC inhibiter, PKA inhibiter (2.3 µM), PD98059 (2.0 µM) a MAP kinase inhibitor and Herbimycin (12 µM) a tyrosine kinase inhibitor (Figure 3). Results were expressed as a percentage of the mean control value. There was a 34% reduction in phosphorylation of the 20 KDa protein in mitochondria that had been incubated with chelerythrine, however, this was not found to be significant (p = 0.1). In mitochondria that were incubated with PKA inhibitor there was a 43% reduction in 20 kDa band intensity that was at the border of the significance threshold (p = 0.05). In mitochondria incubated with PD98059 there was a 29 % reduction in signal from the 20 kDa protein and this was not significant (P = 0.2). Herbimycin reduced the band intensity of the 20 kDa band by 83%, a reduction that was found to be significant (p = 0.01).

Tyrosine Phosphorylation Phosphorylation at tyrosine residues was analysed using a commercial antibody and Western blot (Figure 4). Mitochondria displayed clearly discernible bands with molecular weights at 105, 85, 59, 48, 20 and 16 kDa. Band intensities were compared between heavy mitochondria and light mitochondria preparations from three different placentae. Figure 4a shows a representative autoradiograph depicting bands of tyrosine phosphorylated proteins in heavy and light mitochondria.

Figure 4b shows that heavy and light mitochondria did not display any significant difference in the expression of the 105, 85, 59, 48 kDa proteins. The 20 and 16 kDa phospho-tyrosine proteins, however, displayed bands with significantly higher intensities (both p< 0.001).

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Immunoprecipitation with phosphotyrosine antibodies Immunoprecipitation of 32P labelled mitochondrial protein consistently resulted in a major band at 20 kDa (Figure 5). A very faint band at 22 kDa was also observed. In samples from 2 of the 4 placentae utilised (placentae A and C) bands at 85 kDa were discernible, however, this was not a reproducible result as the other two placentae sampled did not display the 85 kDa band.

Protein kinase activity PKC activity was found in mitochondria to be 31 +/- 9 % of that found in whole placental tissue (Figure 6). This constituted a significant difference in the activity of this kinase in isolated mitochondria as compared with whole placental tissue (p=0.01). PKA activity, however, was not significantly different (p = 0.27) at 83 +/- 12 % that found in whole tissue (Figure 7).

Discussion

The data presented in Figure 1 demonstrates that incubation of mitochondria from human placenta with [γ-32P]-ATP results in newly phosphorylated and radio-labelled proteins. Because [γ-32P]-ATP was added after the isolation of mitochondria the labelled proteins shown in Figure 1 do not display phosphorylation that had occurred prior to mitochondrial isolation. A protein with a molecular weight of 20 kDa was predominantly apparent and this protein may be present at higher concentrations in the mitochondria as compared to the 85 and 38 kDa proteins which displayed fainter detection. Alternatively, the 20 kDa protein may contain more serine, threonine and tyrosine sites available for phosphorylation as compared to 85 kDa and 38 kDa. In later studies it will be interesting to determine whether the phosphoproteins in human

placental

mitochondria

display

homology

with

mitochondrial

phosphoproteins found in rat hepatocytes (Vargas et al., 1982) and bovine heart (Papa et al., 1996).

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Figure 2 shows that 0.5 µCi of [γ-32P]-ATP is an optimal amount to label 20 µg of protein. Using 0.1 µCi [γ-32P]-ATP resulted in a signal from the 20 kDa proteins which was less than half as intense than that obtained with 0.5 µCi [γ-32P]-ATP.

Figure 3 shows the effects of various protein kinase inhibitors on the labelling of the 20 kDa mitochondrial protein with [γ-32P]-ATP. Chelerythrine, and PD98059 resulted in reductions of labelling that were not significant. Treatment with PKA inhibitor resulted in a decrease that was at the borderline of a significant value (p=0.05). Incubation of mitochondria with Herbimycin caused an 83 % reduction in phosphorylation of 20 kDa that was significant (p=0.01). This result suggests that the major kinase which is responsible for the phosphorylation of 20 kDa is a tyrosine kinase.

One of the earliest findings of tyrosine phosphorylated proteins in mitochondria was reported

by

Hakansson

and

Allen

(1995)

who

demonstrated

tyrosine

phosphorylation of a 37 kDa protein in pea mitochondria. In the present study we report that light and heavy mitochondria from human placenta displayed a number of tyrosine phosphorylated proteins (Figure 4). Unlike labelling with [γ-32P]-ATP in isolated mitochondria (Figure 1), labelling with tyrosine phosphate antibody is able to display proteins that were phosphorylated prior to the isolation of mitochondria. Additionally, the proteins that were detected by tyrosine phosphate antibody could have included proteins that were substrates for cytosolic kinases whereas cytosol was removed prior to labelling with [γ-32P]-ATP. It is not therefore surprising that there were more proteins labelled by tyrosine phosphate antibody (Figure 4) than by [γ-32P]-ATP (Figure 1). The 20 and 85 kDa proteins labelled by the antibody to tyrosine phosphate may be the same, however, as the 20 and 85 kDa proteins that were labelled with [γ-32P]-ATP.

The intensities of the 105, 85, 59 and 48 kDa tyrosine phosphate protein bands in heavy mitochondria were not significantly different than those displayed in light mitochondria. This indicates that the concentration of these proteins or their tyrosine phosphorylation status does not play a part in functions that are peculiar to 10

either heavy or light mitochondria. In contrast, the intensity of labelling of the 20 and 16 kDa bands was significantly greater in heavy mitochondria. This suggests that their may be functions of the placental heavy mitochondria that need a higher concentration of the 20 and 16 kDa tyrosine phosphate proteins as compared to light mitochondria as discussed later.

Herbimycin has been shown to cause an increase in number of mitochondria with increased mass in a human colon carcinoma cell line (Mancini et al., 1997). This indicates that tyrosine kinases can alter the physiology of the mitochondria presumably through phosphorylation of a mitochondrial protein. In future studies the effects of herbimycin could be investigated on the number and structure of mitochondria found in human placenta. Such studies would characterize the role of tyrosine phosphorylated proteins in this organelle.

Immunoprecipitation of the

32

P labelled 20 kDa protein with phosphotyrosine

antibody indicates that this protein is phosphorylated on tyrosine residues (Figure 5). It would also appear that the 20 kDa protein labelled with [γ-32P]-ATP is the same protein as that labelled using an anti tyrosine phosphate antibody in Western blot detection (Figure 4). It should be kept in mind that the tyrosine phosphate antibodies could have different affinities for the 20, 22, 38 and 85 kDa proteins. Therefore when the 20, 22, 38 and 85 kDa proteins are selected from a solution by the tyrosine phosphate antibodies they may be present in different ratios as compared to a solution prior to immunoprecipitation. The 85 kDa protein was detected in two out of the four placentae analysed (Figure 5). This may indicate that there is an individual variation in the amount of tyrosine phosphorylation on the 85 kDa protein. Alternatively or additionally, the amount of the expression of the 85 kDa protein may vary between individuals.

While it appears that the majority of phosphorylation on 20 kDa was present due to tyrosine residues it was also decided to determine whether the human placental mitochondria contained serine/threonine kinase activity. PKC activity was significantly lower (p=0.01) at only 28% as high as that found in whole cells. On the 11

other hand PKA activity in placenta was present at 83% the level found in whole cells, a difference which was not significant (p=0.27). It appears therefore that PKA activity is present in the mitochondria at a level comparable to other portions of the cell (relative to protein content) where it is known to perform important functions. Whether it is performing an important duty in the mitochondria is unknown at this stage. Additionally, it should be kept in mind that kinases operating in the mitochondria may be phosphorylating proteins in the mitochondria or proteins external to the organelle.

PKA activity has also been demonstrated in mitochondria from cattle (Papa et al., 1999) and in an invertebrate (Valejo, 1999). Huang (1999) et al., have described a dual purpose AKAP which may be binding PKA to the endoplasmic reticulum and the mitochondria. Papa et al. (1999) propose that the function of PKA in mitochondria from mammals may be to regulate mitochondrial enzymes and transporters. The roles that such a system plays in the development and function of the placenta could provide an interesting field of study.

The mitochondrion has many physiological functions in the development, growth, and function of the eukaryotic cell. In addition to providing ATP, mitochondrial functions can include, vital roles in the synthesis of steroid hormones (Thomson, 1998b), the generation of free radical species and participation in apoptosis (Wallace, 1999). It is now believed that the functions of the mitochondria can be modulated by the host cell. For example, as a result of protein hormone stimulation at the cell surface the mitochondria in steroidogenic tissue can be induced to convert cholesterol to pregnenolone. This step is catalysed by cytochrome P450 side chain cleavage (P450scc) which is the first step in the synthesis of all steroid hormones (reviewed in Thomson, 1998b, Stocco, 2000). An exciting challenge now exists therefore to investigate whether the mitochondrial contains phosphorylating systems are regulating processes such as apoptosis and steroidogenesis. Some work on the physiological roles of mitochondrial protein phosphorylation has recently emerged. Synthesis of the mitochondrial steroidogenic acute regulatory protein (StAR) is necessary for full acute stimulation of steroidogenesis in adrenal and 12

gonadal tissue (reviewed in Thomson, 1998b, Stocco, 2000). This protein is often present as a phosphoprotein and phosphorylation on a serine residue appears to modulate its steroidogenic activity (Arakane et al., (1997). It will be important to determine whether mitochondria in the adrenal contain intrinsic mitochondrial PKA and tyrosine kinases which appear from this current study to be present in the placenta. It will also be valuable to find out whether StAR can be phosphorylated by such mitochondrial kinases.

While the placenta does not express StAR it does express a protein with sequence homology, MLN64 (Watari et al., 1997). MLN64 is observed in a 50 kDa form as well as 42 kDa and 33 kDa forms which may be processing products of the 50 kDa sequence (Watari et al., 1997). It is not known at this point, however, whether the phosphorylation status of MLN64 is crucial for activity of this protein. Nonetheless, it will be interesting in future studies to determine whether MLN64 can be phosphorylated by mitochondrial or cytosolic kinases.

It

would

appear

that

during

differentiation

of

cytotrophoblast

into

syncytiotrophoblast cellular signals are sent to the mitochondria and it is interesting that light mitochondria showed less tyrosine phosphorylation of the 16 and 20 kDa proteins as compared to heavy mitochondria (Figure 4). Light mitochondria are characteristic of syncytiotrophoblast (Martinez, 1996). The differentiation of cytotrophoblast into syncytiotrophoblast may be modulated by a variety of signalling factors including estrogen (Pepe and Albrecht, 1999), protein hormones, cytokines, growth

factors

and

glycoproteins

(Bischof,

2000).

Additionally,

the

syncytiotrophoblast has increased mitochondrial P450scc activity and therefore increased steroidogenic activity as compared to the cytotrophoblast (Strauss et al., 1996). It will be important to determine in future whether the phosphorylation status of mitochondrial proteins on tyrosine, serine or threonine residues plays a part in syncytiotrophoblast differentiation and the activation of the steroidogenic machinery in the placental mitochondrion.

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For the mitochondrion to be receptive to the metabolic needs of the cell it would be expected that there are mechanisms which allow messages to be transmitted from the cytoplasm to the mitochondrion. Because the mitochondrion can contain kinase activity and protein substrates, it would appear that messages could be conveyed to the mitochondrion via protein phosphorylation systems. It will be of great interest to determine in future studies the roles of the 20 kDa phosphoprotein in the physiology of the placental mitochondrion.

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Figures Figure 1. Mitochondrial proteins from human placenta phosphorylated with [γ-32P]ATP.

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Figure 2. Dose response of labelling mitochondrial proteins with various amounts of [γ-32P]-ATP. Samples containing 20 µg of protein were incubated with [γ-32P]-ATP for 10 min. (a) Lanes 1, 2, 3, 4, 5, and 6 show the difference in phosphorylation when placental mitochondria is incubated with 0.5 µCi, 0.2 µCi, 0.1 µCi, 0.06 µCi, 0.005 µCi and 0.0005 µCi [γ-32P]-ATP respectively. (b) Area under the curve for mitochondrial proteins. Similar results obtained in three separate experiments using different placentae.

a)

1

2

3

4

5

6

31

b)

18

16

Figure 3. The effects of various kinase inhibiters on protein phosphorylation of mitochondrial proteins. Equal amounts (5 µg) of mitochondrial proteins were incubated for 10 minutes with 5 µCi [γ-32P]-ATP in the presence or absence of one of the protein kinase inhibitors. (a) Lane 1, 2, 3, 4, and 5 contained mitochondria incubated without inhibitor, with chelerythrin, with PKA inhibitor, with PD98059, and with herbimycin respectively. (b) Scion Image was used to measure the density of phosphorylation. Values represent the mean + SD of triplicate samples.

a) KDa

1

2

3

4

5

18

b)

17

Figure 4. Tyrosine phosphorylation in proteins from heavy and light mitochondria. a) autoradiograph, b) densitometry from experiments performed using three different placentae.

a) 20 85 42 31

18 b)

light

heavy

18

Figure 5. Tyrosine phosphate antibody immunoprecipitation of mitochondrial protein labelled with 32P. Immunoprecipitated proteins were analysed by SDS PAGE and autoradiography. The experiment was performed using four different placentae with duplicate samples from each placenta as follows, lanes 1, 2 placenta A, lanes 3,4 placenta B, lanes 5,6 placenta C, lanes 7,8 placenta D.

1

2

3

4

5

6

7

8

20 85 42 31

18 7

19

Figure 6. The activity of PKC in whole placental tissue and mitochondria. The activity of protein kinase C was tested on placental mitochondria and whole placental extract using a non radioactive protein kinase assay kit. Values are the mean + SD of triplicate samples from 3 different placentae.

tissue

20

Figure 7. The activity of PKA in whole placental tissue and mitochondria. The activity of protein kinase A was tested on placental mitochondria and whole placental extract using a non radioactive protein kinase assay. Values are the mean + SD of triplicate samples from 3 different placentae.

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Acknowledgments This work was performed with support from a University of Western Sydney Research Grant. We wish to thank the staff of the Delivery Suite, Westmead Hospital, NSW, for their kind assistance with the collection of placentae.

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