Phosphorylation Of Plasma Membrane Proteins Research Articles

Detection of Phosphorylation on Immunoprecipitates from Total Protein Extracts of Arabidopsis thaliana Seedlings.

Phosphorylation is a versatile posttranslational modification that can regulate the localization, stability, and conformation of proteins; protein-protein interactions; and enzyme activities. Phosphorylation of plasma membrane proteins, for example, can serve as recognition signals for ubiquitin ligases and hence can trigger its endocytic degradation. Key determinants of protein phosphorylation are kinases and phosphatases that are spatiotemporally regulated to phosphorylate or dephosphorylate specific target proteins. To understand the dynamics and regulatory mechanisms of protein phosphorylation, it is essential to analyze the phosphorylation status of the proteins and identify phosphorylation sites as well as the modifying enzymes. In this chapter, we describe methods that can be used for the detection of phosphoproteins that are immunoprecipitated from Arabidopsis total extracts.

Phosphorylation‐dependent Trafficking of Plasma Membrane Proteins in Animal and Plant Cells

In both unicellular and multicellular organisms, transmembrane (TM) proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane (PM)-localized TM proteins (PM proteins), especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED (PIN) carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples.

TiO2-Based Phosphoproteomic Analysis of the Plasma Membrane and the Effects of Phosphatase Inhibitor Treatment

Phosphorylation of plasma membrane proteins frequently initiates signal transduction pathways or attenuate plasma membrane transport processes. Because of the low abundance and hydrophobic features of many plasma membrane proteins and the low stoichiometry of protein phosphorylation, studies of the plasma membrane phosphoproteome are challenging. We present an optimized analytical strategy for plasma membrane phosphoproteomics that combines efficient plasma membrane protein preparation with TiO(2)-based phosphopeptide enrichment and high-performance mass spectrometry for phosphopeptide sequencing. We used sucrose centrifugation in combination with sodium carbonate extraction to achieve efficient and reproducible purification of low microgram levels of plasma membrane proteins from human mesenchymal stem cells (hMSCs, 10(7) cells), achieving more than 70% yield of membrane proteins. Phosphopeptide enrichment by titanium dioxide chromatography followed by capillary liquid chromatography-tandem mass spectrometry allowed us to assign 703 unique phosphorylation sites in 376 phosphoproteins. Our experiments revealed that treatment of cell cultures with three different types of protein phosphatase inhibitors produces distinct phosphopeptide populations and an increase of 10-40% of the number of detected and sequenced phosphoserine, phosphothreonine and phosphotyrosine containing peptides. In summary, our analytical strategy enables functional phosphoproteomic analysis of stem cell differentiation and cell surface biomarker discovery using very low amounts of starting material.

Capacitation Induces Tyrosine Phosphorylation of Proteins in the Boar Sperm Plasma Membrane

Capacitation (activation) of mammalian spermatozoa is accompanied by protein phosphorylation, elevation of the intracellular calcium concentration and an increased plasma membrane fluidity. The subcellular localization of tyrosine phosphorylation during capacitation have not yet been elucidated. The aim of this study was to investigate whether boar sperm capacitation induces tyrosine phosphorylation of plasma membrane proteins. Capacitation induced tyrosine phosphorylation of 3 proteins (27, 37, and 40 kDa), which coincided with an increase in the plasma membrane fluidity. The importance of the induced tyrosine phosphorylation in sperm binding to the zona pellucida and the induction of the acrosome reaction is discussed.

Rapid blue-light-induced phosphorylation of plasma-membrane-associated proteins in wheat

When the microsomal membranes isolated from 4-day-old etiolated wheat seedlings are irradiated in vitro with blue light and subjected to phosphorylation with [γ- 32P]ATP in vitro, the polypeptides of 110, 102, 82 and 70 kDa are heavily phosphorylated. The induction of phosphorylation is extremely rapid as it can be triggered by even 5 sec blue light irradiation, although maximal response is obtained with 15 sec blue irradiation. The response is observed most strongly in the microsomal fraction isolated from the highly photosensitive tip portion of the seedlings, and its intensity is higher in the leaf than in the tubular coleoptile fraction. The aqueous two-phase partitioning of the microsomal fraction has shown that all these four blue-light-responsive phosphopolypeptides are associated with the plasma membrane. The kinase involved in phosphorylating these polypeptides in vitro is sensitive to staurosporine. The presence of blue-light-responsive phosphopolypeptides in both the coleoptile sheath and the leaf encased inside, and the kinetics of induction of this response indicate that the phosphorylation of plasma membrane proteins may be one of the early steps in blue-light-elicited signal transduction chain, associated not only with phototropism but perhaps also with low-fluence blue responses in general.

Tyrosine protein phosphorylation in plasma membranes of rat kidney cortex.

The endogenous tyrosine protein kinase activity (TPKA) associated with brush-border (BBM) and basolateral (BLM) membranes of rat kidney cortex was studied with an anti-phosphotyrosine monoclonal antibody (PY20). Distinct major phosphotyrosine-containing proteins were associated with BBM (50, 54, and 120 kDa) and BLM (37, 90, 130, and 170 kDa). For both plasma membranes, tyrosine phosphorylation leveled off after 10 min of incubation. Endogenous phosphotyrosine-specific protein phosphatases (PT-Pases) were active in both membranes, since the presence of sodium vanadate or ammonium molybdate, which are inhibitors of PTPases, was essential to detect endogenous phosphorylation. Substrates and/or tyrosine protein kinases (TPKs) seem to be differently distributed in these plasma membranes, since phosphorylation of endogenous substrates in BLM and BBM was differently sensitive to competitive inhibitors of TPKs. Moreover, insulin- and insulin-like growth factor I-stimulated tyrosine phosphorylation of a 90-kDa substrate was only observed in solubilized BLM proteins. However, similar p60v-src-related TPKs appear to be present in the BBM and BLM, since an antibody raised against p60v-src recognized proteins of 52, 58, and 75 kDa by immunoblotting and could immunoprecipitate the TPKs associated with both plasma membranes. These data provide evidence that the endogenous tyrosine protein phosphorylation observed in the BLM is catalyzed by nonreceptor TPKs as well as receptor TPKs, whereas that observed in the BBM is exclusively due to nonreceptor TPKs.

Phytochrome-mediated light signal transmission to the phosphorylation of proteins in the plasma membrane and the soluble fraction of etiolated pea stem sections

The excised third internodes of etiolated pea seedlings were irradiated with red light (660 nm) for 40 s changing the intensity from 0, 10, 50 and 100 μmol m −2 s −1. The tissue was ground in a buffer and the plasma membrane was purified. The phosphorylation of plasma membrane proteins with molecular masses of 15, 50, 70 and 120 kDa from adenosine 5′[γ- 32P]triphosphate ([γ- 32P]ATP) at 0 °C for 7 s increased with increasing fluence rate of red light from 10 to 50 μmol m −2 s −1. However, the phosphorylation of these proteins was less enhanced at red-light fluence rate of 100 μmol m −2 s −1. Successive irradiation of the third internode by red (100 μmol m −2 s −1) for 20 s and far-red light (730 nm; 4μmol m −2 s −1) for 30 s reversed the phosphorylation of the 15 kDa protein. The 32P phosphoryl group of the 15 kDa protein which was received from [γ- 32P]ATP at 0 °C for 7 s was removed efficiently by subsequent addition at 0 °C for 5 s of cold 10 −6 M adenosine 5′-diphosphate or guanosine 5′-diphosphate and also 10 −5 M ATP or guanosine 5′-triphosphate. The phosphorylation of the 15 kDa protein in the soluble fraction showed a similar light response to that associated in the plasma membrane.

Effects of polyamines on in vitro phosphorylation of soluble and plasma membrane proteins in tobacco, cucumber and Arabidopsis thaliana

Polyamines are essential for growth and development in prokaryotic and eukaryotic organisms. In this study, we report the effect of cadaverine, putrescine, spermidine and spermine on the phosphorylation of soluble and plasma membrane proteins of tobacco, cucumber and Arabidopsis thaliana. Except for the enhanced phosphorylation of several cucumber soluble proteins, cadaverine had little or no effect on the phosphorylation of soluble and plasma membrane proteins of the three plants. In tobacco, putrescine increased the phosphorylation of some soluble proteins and had no effect on the phosphorylation of plasma membrane proteins, whereas spermidine and spermine decreased the overall phosporylation of soluble proteins but enhanced the phosphorylation of three plasma membrane proteins with molecular weights of 16.5, 34 and 60 kDa. Spermidine had a stronger effect on the phosphorylation of tobacco plasma membrane proteins than spermine and had concentration optimum of ca. 6 mM. Putrescine, spermidine and spermine enhanced the phosphorylation of some soluble proteins from A. thaliana and cucumber and decreased the phosphorylation of others. The three polyamines enhanced the phosphorylation of many plasma membrane proteins. Polyamines increased serine and threonine phosphorylation of plasma membrane proteins prepared from tobacco.

Atrial natriuretic factor inhibits autophosphorylation of protein kinase C and A 240-kDa protein in plasma membranes of bovine adrenal glomerulosa cells: involvement of cGMP-dependent and independent signal transduction mechanisms.

To clarify the intracellular signalling mechanisms of atrial natriuretic factor (ANF), we studied its effect on protein phosphorylation in plasma membranes of bovine adrenal cortical cells. ANF (1 x 10(-7) M) inhibited phosphorylation of the 78-kDa protein kinase C (PKC) and a 240-kDa protein in specific manner. In parallel experiments, cGMP (0.5 mM) inhibited phosphorylation of only the 78-kDa PKC but it did not affect phosphorylation of the 240-kDa protein. Phosphorylation of the 78-kDa PKC was enhanced in a Ca(2+)-/phospholipid-dependent manner. However, after prolonged preincubation of plasma membranes with Ca2+ (0.5 mM), the incorporation of 32P-radioactivity rapidly decreased in the 78-kDa PKC and subsequently increased in the 45- and 48-kDa protein bands due to Ca(2+)-dependent proteolytic degradation of 78-kDa PKC. Polyclonal antibodies against brain PKC were used to immunoblot and immunoprecipitate the 78-kDa PKC. Preincubation of plasma membranes with Ca2+ for varying times, followed by immunoblotting revealed a gradual loss of the immunoreactive 78-kDa PKC band in a time-dependent manner. Immunoprecipitation of phosphorylated 78-kDa PKC in plasma membranes showed that its phosphorylation was significantly inhibited in the presence of ANF as compared to control membranes, phosphorylated in the absence of ANF. The results in this present study document a new signal transduction mechanism of ANF at molecular level which possibly involves dephosphorylation of the 78-kDa PKC and a 240-kDa protein in a cGMP-dependent and -independent manner in bovine adrenal glomerulosa cell membranes.

Ethanol induced alterations in plasma membrane protein phosphorylation of neurons and astrocytes.

The endogenous protein phosphorylation patterns in plasma membranes of bulk isolated neurons and astroglia from control and chronic ethanol treated rats have been investigated. Chronic ethanol treatment resulted in increased phosphorylation of specific proteins with molecular weights 116, 63 and 60 kDa in both neurons and astrocytes. These proteins were further resolved by 2-DE and the analysis suggested an increased phosphorylation of congruent to 10-15 proteins, of which 116 kDa protein is phosphorylated to a higher extent by ethanol. Further, decreased phosphorylation was noticed in D-95 and D-63 proteins in neurons and D-78 and D-54 proteins in astrocytes. Alkali stability experiments for identifying the phosphoamino acid involved in phosphorylation of 116, 63 and 60 kDa proteins suggested that tyrosine and threonine are not involved and probably serine is the likely site for phosphorylation during chronic ethanol treatment. The phosphorylation of specific membrane proteins during chronic ethanol treatment might contribute to ethanol evoked cellular dysfunction.

Ca 2+ ions and lysophospholipids activate phosphorylation of different proteins in plasma membranes and tonoplast

Summary Protein phosphorylation and regulation by phosphorylation of transport ATPases was studied in highly purified plasma membrane and tonoplast vesicles isolated from etiolated zucchini ( Cucurbita pepo L.) hypocotyls. Plant lysophosphatidylcholine and the chemically similar animal lipid platelet-activating factor activated H + transport activity of plasma membrane ATPase in vitro . In plasma membrane vesicles, purified by two-phase partitioning and subsequent free-flow electrophoresis, both lipids activated a membrane-associated protein kinase that phosphorylated a 97 kD polypeptide. A 97 kD polypeptide also gave a western blot signal generated by an antibody prepared against plasma membrane H + -ATPase. The phosphoprotein and the western blot signal were absent in tonoplast fractions purified by free-flow electrophoresis. In contrast, the B subunit of the tonoplast H + -ATPase was identified by western blot in those fractions but could not be found in plasma membrane fractions, corroborating membrane identity. When Ca 2+ -dependent and lysophospholipid-dependent protein phosphorylation in plasma membranes and in tonoplast were compared it was found for both membranes that the phosphorylation of several polypeptides was Ca 2+ -dependent but lysophospholipid-independent. These phosphoproteins were different in plasma membranes and tonoplast. Ca 2+ is an established second messenger in plants, and lysophospholipids are proposed as potential signal mediators activating membrane-associated protein kinase(s), Our data indicate that they could be distinguished by their substrates. A function of both plasma membrane and tonoplast in plant signal transduction pathways is indicated.

Evidence against a role for alkaline phosphatase in the dephosphorylation of plasma membrane proteins: Hypophosphatasia fibroblast study

A major impasse to understanding the physiologic role(s) of alkaline phosphatase (ALP) is uncertainty as to its natural substrates. Various in vitro studies have led other investigators to suggest that ALP functions as a plasma membrane phosphoprotein phosphatase, consistent with our demonstration of ecto-topography of ALP in a variety of cell types. Thus, we compared the phosphorylation of plasma membrane proteins from control fibroblasts to those from profoundly ALP-deficient fibroblasts of hypophosphatasia patients. Fibroblasts from 3 controls and 3 hypophosphatasia patients (ALP activity < 4% of control) were biosynthetically labeled with 32Pi for 2 h. 32P incorporation into total trichloroacetic acid (TCA)-precipitable material was not significantly different in control and patient cells. Plasma membranes were prepared from these cells by hypotonic shock, solubilized, and subjected to two-dimensional (2-D) gel electrophoretic separation. Video densitometric analysis of silver-stained 2-D gels failed to reveal any consistent difference in the protein profile between patient vs. control fibroblasts (i.e., unique species, altered pls, or increased abundance). Autoradiography of individual 2-D gels demonstrated 63 plasma membrane phosphoproteins with molecular weights ranging from 15 to 152 kDa and predominantly acidic pls. Although several of these phosphoproteins appeared to have had donor-specific labeling, none was unique or especially abundant in the hypophosphatasia group. Thus, in ALP-deficient fibroblasts, normal incorporation of 32P into total cellular protein and into all identifiable plasma membrane phosphoproteins indicates that ALP does not modulate the phosphorylation of plasma membrane proteins.

Oligosaccharide signaling in plants. Specificity of oligouronide-enhanced plasma membrane protein phosphorylation.

The in vitro phosphorylation by [gamma-32P]ATP of a 34-kDa plasma membrane-associated protein (pp34) from tomato and potato is strongly enhanced in the presence of alpha-1,4-D-polygalacturonic acid (PGA) fragments (Farmer, E. E., Pearce, G., and Ryan, C. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1539-1542) that activate the expression of defensive and developmental genes in plant tissues. [gamma-35S]ATP, but not [gamma-35S]GTP, has now been found to strongly label pp34 in the presence of the PGA fragments. PGA-enhanced phosphorylation of pp34 is at one or more threonine residue(s) and therefore is the product of a serine/threonine kinase. alpha-1,4-L-Polyguluronic acid (PGU) enhances thiophosphorylation of pp34, but is less effective than PGA. beta-1,4-D-Polymannuronic acid (PMA) is inactive. In vivo synthesis of proteinase inhibitors in tomato leaves in response to PGA, PGU, and PMA parallels enhancing activities in in vitro phosphorylation assays. The minimum oligogalacturonide lengths that enhance in vitro thiophosphorylation of pp34 are about 14-15 residues, which are near the minimum sizes of uronides required to elicit a variety of localized defensive and developmental responses in plants. The lengths of biologically active galacturonic acid oligomers are of the same length that form strong intermolecular complexes in solution with Ca2+. Uronide-Ca2+ complexes are proposed to be the active molecular species that initiate the signal transduction pathways regulating uronide-responsive genes.

Phosphorylation by Inorganic Phosphate of the Plasma Membrane H+-ATPase from Red Beet (Beta vulgaris L.)

The phosphorylation of plasma membrane proteins from red beet (Beta vulgaris L.) by radioactive inorganic phosphate was studied. Only few proteins were phosphorylated, among them was one polypeptide with an apparent molecular weight of about 100,000. The phosphorylation of this protein was decreased when orthovanadate was present in the reaction mixture, or when the phosphorylated protein was treated with hydroxylamine. These facts suggest that this protein is a transport ATPase which is phosphorylated in a carboxyl group during the catalytic cycle. This protein was identified immunologically as the plasma membrane H(+)-ATPase. The phosphorylation level of this enzyme was enhanced by dimethyl sulfoxide, whereas potassium ions did not have a significant effect on this level unless ATP was present. ATP stimulated the phosphorylation by inorganic phosphate. This stimulation was more apparent in the presence of potassium ions.

Stimulation of protein phosphorylation by atrial natriuretic factor in plasma membranes of bovine adrenal cortical cells

Atrial natriuretic factor (ANF) rapidly enhanced phosphorylation of plasma membrane proteins of bovine adrenal cortical cells. Pretreatment of the membranes with ANF (1 x 10(-8)M to 1 x 10(-7)M) resulted two- to four-fold in an incorporation of 32p-radioactivity from [gamma -32p]ATP as compared to the controls. The guanosine 3', 5' monophosphate (cGMP) which has been considered a second messenger of ANF also enhanced the phosphorylation of several proteins which were stimulated by ANF. However, the phosphorylation of certain proteins was stimulated differentially only by either ANF or cGMP. These results suggest that ANF-induced protein phosphorylation may play a role in transmembrane signalling pathway involving other second messenger(s) in addition to cGMP during the biological action of ANF.

Specific phosphorylation by calcium-egta complex of a 75kda human tumor plasma membrane protein (pp75)

1. 1. The major functional role played by phosphorylation of plasma membrane proteins in the biological properties of tumor cells suggests that identification of protein kinases and their substrates will contribute to our understanding of the molecular basis of the malignant process and of the aberrant behavior of tumor cells. 2. 2. The present study has investigated the phosphorylation of surface proteins of human tumor cells. Incubation of plasma membranes isolated from cultured human melanoma cells with [γ- 32P]ATP in the presence of Ca 2+ and ethylene-bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) resulted in specific phosphorylation of serine and threonine residues on a 75kDa protein (pp75). 3. 3. Neither Ca 2+ or EGTA alone, nor any other divalent metal ion tested could induce phosphorylation of pp75. 4. 4. The phosphorylation of pp75 was directly dependent upon the presence of non-ionic detergents, and was influenced by length of incubation and concentration ratio of Ca 2+ and EGTA. 5. 5. Incubation of isolated plasma membranes with [γ- 32P]ATP in the presence of Ca 2+ and EGTA and immunochemical analysis by Western blotting with an anti pp75 xenoantiserum detected the pp75 in human melanoma, neuroblastoma, ovarian carcinoma and lymphoid T cells and fibroblasts but not in B-lymphoid cells, renal carcinoma cells, peripheral blood lymphocytes and splenocytes. 6. 6. These results suggest the presence of a new class of plasma membrane bound protein kinases activated by chelated calcium and differentially expressed in normal and transformed human cells.

Rapid Changes in Plasma Membrane Protein Phosphorylation during Initiation of Cell Wall Digestion

Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with [gamma-(32)P]ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M(r) 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M(r) 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M(r) 22,000. These effects appeared not to be due to nonspecific protease activity and neither in vivo nor in vitro exposure to driselase caused a significant loss of Coomassie blue-staining bands on the gels of the isolated plasma membranes. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic (32)P also showed a response to the Driselase treatment. An enzymically active driselase preparation was required for the observed responses.

Endogenous hyperphosphorylation in plasma membrane from an ascites hepatocarcinoma cell line.

Plasma-membrane-bound kinases of AS-30D ascites from transplantable rat hepatocarcinoma were shown to extensively catalyze the phosphorylation of plasma membrane proteins and membrane lipids, using [gamma-32P]ATP or [gamma-32P]GTP as a phosphate donor. In contrast, plasma membranes from normal adult rat liver or fast-growing regenerating liver (24 h after partial hepatectomy) produce significantly less activity for protein phosphorylation and little phosphorylation of the lipids. However, neonatal (24 h old) rat liver plasma membrane preparations show levels of phosphorylation of proteins and lipids intermediate between those in the tumor cell line and normal adult plasma membrane preparations. Phosphatidic acid was identified as one of the 32P-labelled lipids in the tumor plasma membrane chloroform-methanol (2:1, v/v) extract. Phosphorylation of protein was not affected by cAMP or cGMP. However, calcium ion (in the presence or absence of calmodulin) significantly modifies the 32P labelling of a series of proteins in normal tissue but has little effect with the neoplastic preparations. Some plasma membrane proteins were capable of nucleotide binding, instead or in addition to being phosphorylated. Finally, the presence of membrane-bound phosphoprotein phosphatase(s) was also demonstrated in all the preparations examined by means of chase experiments with nonlabelled ATP or GTP, and (or) by the use of the phosphoprotein phosphatase inhibitor, orthovanadate.

New signal transduction mechanisms of atrial natriuretic factor: Inhibition of phosphorylation of protein kinase C and a 240 kDa protein in adrenal cortical plasma membrane by cGMP dependent and independent mechanisms

The effects of synthetic atrial natriuretic factor (ANF) on the state of protein phosphorylation in plasma membranes of bovine adrenal cortex have been studied in vitro . ANF (1×10 −8M – 1×10 −7M) specifically inhibited the phosphorylation of two distinct proteins of 78 kDa and 240 kDa. Immunoblotting with specific antiserum to protein kinase C produced evidence that 78 kDa protein is most likely the protein kinase C whose phosphorylation is inhibited by both ANF and cGMP. However, cGMP did not affect the phosphorylation of 240 kDa protein, indicating a new cGMP-independent mechanism of ANF action in the adrenal, which is compatible with the lack of action of cGMP and its analogs in ANF-induced inhibition of aldosterone secretion from adrenal cortex. The inhibition of phosphorylation of putative protein kinase C by ANF or cGMP indicates a hitherto unknown signal transduction mechanism of ANF.

Endogenous phosphorylation of proteins and phosphatidylinositol in the plasma membranes of a human astrocytoma

Incubation of plasma membrane preparations from several tissues with [γ- 32P]ATP resulted in the phosphorylation of phosphatidylinositol as well as of proteins. The presence of an active phosphatidylinositol kinase in these membranes was indicated by equal or greater incorporation of 32P into phosphatidylinositol phosphate than into proteins. Phosphorylation of endogenous protein and lipid substrates by protein and phosphatidylinositol kinases in the plasma membranes of a human astrocytoma was investigated in detail. Maximal protein phosphorylation required the presence of Non-idet-P40 and phosphatase inhibitors (vanadate or fluoride). The rate of protein phosphorylation was greater with Mg 2+ than with Mn 2+, and phosphoserine accounted for 60% of the radioactivity incorporated into proteins. In the presence of Mn 2+, phosphorylation of tyrosine was increased and was equal to that of serine phosphorylation (40%). With one exception, the overall pattern of phosphorylated proteins was similar with either Mg 2+ or Mn 2+. Maximal phosphatidylinositol phosphorylation of the astrocytoma plasma membranes also required detergent and phosphatase inhibitors. However, the enzymatic characteristics of lipid phosphorylation differed from those of protein phosphorylation with respect to divalent cation activation, ATP dependence, and sensitivity to inhibition by p-chloromercuriphenyl sulfonate, quercetin, and nucleoside derivatives. These results suggest that phosphorylation of plasma membrane proteins and phosphatidylinositol is catalyzed by different enzymes. The fact that membrane preparations exhibited phosphatidylinositol kinase activity almost 100,000 times greater than that exhibited by the purified tyrosine kinase of ros gene would exclude this and similar oncogene proteins from making a significant contribution to the overall phosphatidyl-inositol phosphorylation of cell membranes.