Báo cáo khoa học: Identification and characterization of novel PKA holoenzymes in human T lymphocytes pdf

holoenzymes in human T lymphocytesSigurd Ørstavik1, Ane Funderud1, Tilahun Tolesa Hafte1, Sissel Eikvar1,2, Tore Jahnsen2 and Bjørn Steen Ska˚lhegg1 1 Department of Nutrition, Institute

holoenzymes in human T lymphocytes

Sigurd Ørstavik1, Ane Funderud1, Tilahun Tolesa Hafte1, Sissel Eikvar1,2, Tore Jahnsen2

and Bjørn Steen Ska˚lhegg1

1 Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Norway

2 Department of Biochemistry, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Norway

In the absence of cAMP, cAMP-dependent protein

kinase (PKA) is a holoenzyme consisting of two

regu-latory (R) subunits bound together in a dimer, with

one catalytic (C) subunit bound to each R-subunit

Binding of two cAMP molecules to each R subunit

results in a conformational change that promotes

release of active C subunits In mammals, four genes

encode different isoforms of the R-subunits, RIa, RIb,

RIIa and RIIb, and three different genes encode three

C isoforms, Ca, Cb and PRKX [1] The Ca and Cb

isoforms are closely related in protein sequence while

the PRKX is more different from Ca and Cb In

pri-mates, a transcribed retroposon Cc has been identified

However, this gene has never been shown to express a

protein sequence in vivo making the function of Cc

unclear [2] Several splice variants have been identified

for Ca and Cb In the case of Ca, two active isoforms

have been identified and designated Ca1 and Cas Ca1

is expressed in most tissues while Cas is restricted to sperm cells [3,4] So far, 10 different splice variants of

Cb have been identified, as a result of alternative spli-cing of seven different exons encoding the N-terminal part of the Cb protein [5] Whereas the majority of the

Cb splice-variants are expressed in a brain-specific manner [6] the Cb2 splice variant is highly expressed in lymphoid tissues Most C isoforms, including Ca1 and Cb1, have a calculated molecular mass of  40 kDa, and with a lack of isoform-specific antibodies they may be difficult to distinguish In contrast, the Cb2 isoform has a calculated molecular mass of 47 kDa [6] making it more easy to distinguish from other C-sub-units by SDS⁄ PAGE analysis

The high level of Cb2 mRNA observed in lymphoid tissues led us to investigate whether Cb2 protein could

be present in T cells Previously, T cells have been shown to contain mainly RIa (80%) and some RIIa

Keywords

antibodies; cAMP; lymphocytes; protein

kinase A

Correspondence

B Steen Ska˚lhegg, Department of Nutrition,

Institute of Basic Medical Sciences, Faculty

of Medicine, University of Oslo, PO Box

1046, Blindern, N-0316 Oslo, Norway

Fax: +47 2285 1347

Tel: +47 2285 1547

E-mail: bjorn.skalhegg@medisin.uio.no

Website: http://www.uio.no

(Received 2 November 2004, revised 22

December 2004, accepted 13 January 2005)

doi:10.1111/j.1742-4658.2005.04568.x

Cyclic AMP-dependent protein kinase (PKA) is a holoenzyme that consists

of a regulatory (R) subunit dimer and two catalytic (C) subunits that are released upon stimulation by cAMP Immunoblotting and immunoprecipita-tion of T-cell protein extracts, immunofluorescence of permeabilized T cells and RT⁄ PCR of T-cell RNA using C subunit-specific primers revealed expression of two catalytically active PKA C subunits Ca1 (40 kDa) and Cb2 (47 kDa) in these cells Anti-RIa and Anti-RIIa immunoprecipitations demonstrated that both Ca1 and Cb2 associate with RIa and RIIa to form PKAI and PKAII holoenzymes Moreover, Anti-Cb2 immunoprecipitation revealed that Ca1 coimmunoprecipitates with Cb2 Addition of 8-CPT-cAMP which disrupts the PKA holoenzyme, released Ca1 but not Cb2 from the Anti-Cb2 precipitate, indicating that Cb2 and Ca1 form part of the same holoenzyme Our results demonstrate for the first time that various C sub-units may colocate on the same PKA holoenzyme to form novel cAMP-responsive enzymes that may mediate specific effects of cAMP

Abbreviations

C, catalytic subunit of PKA; FITC, fluoresceinisothiocyanate; PKA, protein kinase A; PKI, protein kinase inhibitor; PLC-c1 ⁄ 2, phospholipase

C c1 ⁄ 2; PVDF, poly(vinylidene difluoride); R, regulatory subunit of PKA; TRITC, tetramethyl-rhodamineisothiocyanate.

(10–20%) as regulatory subunits, and Ca and Cb

mRNA [7] In the present study we demonstrated that

T lymphocytes express significant amounts of active

Cb2 protein which together with Ca1 associate with

both RIa and RIIa to form PKAI and PKAII

holo-enzymes in T cells

Results and Discussion

Cb2 is expressed in human T cells

We have previously shown that human T cells express

Ca and Cb mRNA [7] and that human immune tissues

such as spleen and thymus express Cb1 and Cb2

mRNA [6] Total RNA was isolated from human

T cells, the T-cell line Jurkat and NT-2 cells, and

amplified by RT⁄ PCR using Cb1 and Cb2-specific

primers (Fig 1A) This demonstrated the presence of

Cb1 and Cb2 in all cell types examined The Cb2

cDNA encodes a protein with an approximate

mole-cular mass of 47 kDa [6,8,9] To investigate if Cb2

protein is expressed in human T cells we used various

anti-C antibodies on lysates of resting T cells and

Jur-kat cells As shown in Fig 1B, three commercially

available polyclonal antibodies (PKAacat,

anti-PKAbcat and anti-PKAccat) and one monoclonal

antibody (anti-Cmono) were immunoreactive to two protein bands of 40 and 47 kDa, respectively (Fig 1B) All of these antibodies were raised against regions in the C subunit that are conserved and therefore do not differ significantly between the C isoforms (Fig 1C) Based on this we suggested that both the 47 and

40 kDa forms represented C subunit variants It has been well documented that the C subunits Ca1 and Cb1 are expressed as 40 kDa proteins [10] According

to the immunoreactivity and theoretical molecular mass the 47 kDa protein may therefore correspond to the Cb2 splice variant To confirm this, we generated two different antibodies: anti-Cb2(SNO103) was raised against peptide sequences found in the splice variant-specific part of Cb2 and should therefore only react with this isoform; anti-Cb(SNO157) was generated by immunizing a rabbit using a peptide common to all Cb splice variants, but with only weak homology to the Ca-sequence This antiserum should therefore in the-ory recognize all Cb splice variants and not cross-react with Ca (Fig 1C and Experimental procedures) Anti-Cb2(SNO103) recognized the 47 kDa protein band in

T cells and Jurkat cells, while anti-Cb(SNO157) recog-nized a 47 kDa band in T cells and Jurkat cells and a

40 kDa protein band in Jurkat cells As no 40 kDa band could be detected using the anti-Cb(SNO157) in

A

C

T-cell Jurkat NT2 B

Fig 1 Identification of PKA catalytic subunits in human T cells (A) Human T cells express both Cb1 and Cb2 mRNA Total RNA isolated from human T-cells, the T cell line Jurkat and human NT-2 cells were reverse-transcribed (+) and amplified using primers specific for the Cb1 mRNA (upper panel) and Cb2 mRNA (lower panel) Parallel reactions were performed without reverse transcriptase (–) to demonstrate specificity The amplified fragments were separated by agarose gel-electrophoresis Both Cb1 and Cb2 were detected in all three cell types tested, but a significantly weaker signal of Cb1 was detected in T cells, while a significantly weaker signal of Cb2 was detected in NT-2 cells (B) Lysates of human peripheral blood T cells (T) and the Jurkat T cells (J) were subjected to SDS ⁄ PAGE in 12.5% gels, transferred to PVDF membranes and immunoblotted using three commercially available polyclonal antibodies: PKAacat (i), PKAccat (ii) and anti-PKAbcat (iii), and a commercial monoclonal antibody, anti-Cmono (iv) These antibodies all recognized several different protein bands, but two bands of 40 and 47 kDa were common to all The Cb2-specific antibody anti-Cb2(SNO103) (v) recognized the 47 kDa band in T cells A Cb-specific antibody anti-Cb(SNO157) (vi) recognized the 47 kDa band in T-cells, and the 40- and 47 kDa protein bands in Jurkat cells (C) Lin-ear depiction of Ca1, Cb1 and Cb2 amino acid sequence and the localization of the domains ⁄ antigens used to generate the different anti-C antibodies used in (B).

T cells, it suggested that Cb1 is expressed at

nondetect-able levels in human T cells applying the panel of

anti-bodies used here (Table 1) This is consistent with the

fact that a significantly weaker Cb1 signal was

ampli-fied by RT⁄ PCR from T cells when compared to the

signals amplified from Jurkat RNA Based on this, we

concluded that the two major isoforms of the PKA C

subunit in T cells are Ca1 (40 kDa) and Cb2 (47 kDa),

respectively

Cb2 cDNA encodes an active kinase recognized

specifically by anti-Cb2

Previously it has been demonstrated that a cDNA

encoding the bovine Cb2 was not active when

trans-fected into CHO10260 cells [9] This prompted us to

express the human Cb2 splice variant in eukaryotic

cells Expression vectors encoding native full-length

Ca1 (pEFDEST51Ca1) and Cb2 (pEFDEST51Cb2)

under a mammalian promoter were constructed (see

Experimental procedures) and transiently transfected

into 293T cells Post-transfection the cells were lysed,

subjected to SDS⁄ PAGE in 12.5% gels and

immuno-blotted using anti-Cmono Fig 2A (upper panel)

depicts that transfection with pEFDEST51Ca1 yielded

a 40 kDa anti-Cmono immunoreactive protein while

transfection using pEFDEST51Cb2 resulted in a

47 kDa immunoreactive protein The mock-transfected

cells expressed only a 40 kDa C This demonstrated

that human Cb2 cDNA encodes a 47 kDa protein

which is immunoreactive to anti-Cmono To verify the

specificity of the anti-Cb2(SNO103) antibody the

ly-sates were immunoblotted using the anti-Cb2(SNO103)

antibody, as shown in Fig 2A (middle panel) The

anti-Cb2 antibody recognized a 47 kDa band only

pre-sent in the Cb2-transfected cells and not in the Ca1 or

mock transfected cells This demonstrated the

specifici-ty of anti-Cb2(SNO103) in immunoblots because the

40 kDa protein band of cells overexpressing Ca1 and

the 40 kDa protein band of endogenous C were not

detected Furthermore, by monitoring PKA-specific

kinase activity of the various cell lysates using

Kemptide as a substrate a 3.9- and 3.4-fold higher activ-ity was revealed in the extracts of Ca1 and Cb2 trans-fected cells, respectively, as compared to the mock transfected cells This demonstrated that human Cb2 cDNA encodes an active protein kinase capable of phosphorylating a typical PKA substrate Next we immunoprecipitated from the Ca1, Cb2 and mock transfected cell lysates using anti-Cb2(SNO103) in the presence of 1 mm 8-CPT-cAMP The cAMP analogue was included to avoid potential binding of non-Cb2 C-subuints to the R-subunits The resulting precipitates were analysed by SDS⁄ PAGE and immunoblotting using anti-Cmono (Fig 2B) and a PKA-specific kinase assay Anti-Cb2(SNO103) immunoprecipitated a

47 kDa protein only in the Cb2 transfected cells which also demonstrated the specificity of anti-Cb2(SNO103)

in immunoprecipitation assays The kinase activity in the precipitate from Cb2 transfected cells were 15-fold higher than the activity in the precipitate from the Ca1 transfected cells demonstrating that the Cb2 antibody

in combination with PKA kinase assay could be used to detect specifically Cb2 activity To finally conclude that anti-Cb2(SNO103) is specific for Cb2, the Cb2 and mock transfected 293T cells were subjected to immuno-fluorescence We used anti-Cb2(SNO103) and fluoresce-ine isothiocyanate (FITC)-conjugated secondary antibody to detect Cb2 (Fig 2C, upper and lower left panels) and anti-c-tubulin and TRITC-conjugated sec-ondary antibody to detect the centrosome (Fig 2C, upper and lower middle panels) This demonstrated that cells transfected with Cb2 have increased levels of pro-teins immunoreactive to anti-Cb2(SNO103) In addi-tion, this revealed that Cb2 was distributed in the cytosol as well as enriched in the centrosome as judged

by the anti-c tubulin staining when merged with the anti-Cb2 staining and compared to mock transfected cells (Fig 2C, upper and lower right panels) Taken together our results from immunoblotting, immunopre-cipitation, immunofluorescence in addition to measur-ing enzyme activity, enable us to conclude that anti-Cb2(SNO103) specifically recognizes Cb2, and that Cb2 cDNA encode an active protein kinase

Table 1 Antibodies used to identify PKA subunits in T cells at the protein level.

Anti-PKAccat Santa-Cruz biotechnology C-terminal peptide Cc Cross reacts with Ca and Cb Rabbit

no cross-reaction with Ca

Rabbit

Cb2 in T cells associates with RIa and RIIa

Human T cells express the R subunits, RIa and RIIa

[7] RIa is considered the major R subunit in T cells

making up more than 80% of the total R subunit

activity implying that RIIa constitutes only 10–20% of

the activity To compare the subcellular localization of Cb2, RIa and RIIa, immunofluorescence was per-formed using anti-Cb2(SNO103) in combination with anti-RIa (Fig 3A, top panel) and anti-RIIa (Fig 3A, lower panel) As previously described [11] the RIa is located at the membrane⁄ cytosolic area, while the RIIa

A

32

±5

474

±179

10

±6

50 37

Mw

Cβ2

Catalytic activity (pmol ATP/mL/min)

Transfection: Cα1 Cβ2 Mock

B

Anti-Cβ2

Merge

Merge Anti-γ-tubulin

Transfection

Cβ2

Mock

C

Fig 2 The human Cb2 cDNA encodes an active protein kinase and the Cb2 peptide antibody is specific for human Cb2 (A) Human 293T cells were transiently transfected using plasmids encoding Ca1 (Ca1) and Cb2 (Cb2) or the cells were mock transfected (Mock) Cells were lysed and protein extracts subjected to SDS ⁄ PAGE, transferred to PVDF membranes and immunoblotted using anti-Cmono (anti-Cmono, upper panel) and anti-Cb2(SNO103) (middle panel) Lysates were also analysed for PKA-specific kinase activity (lower panel) (B) The lysates from transfected 293T cells were subjected to immunoprecipitation using anti-Cb2, followed by SDS ⁄ PAGE transferred to PVDF membranes and immunoblotted using anti-Cmono (upper panel, B) The same immunoprecipitates were analysed for PKA-specific kinase activity (lower panel, B) (C) 293T cells were either transfected with Cb2 (upper panels, Cb2) or mock transfected (lower panels, Mock) and subjected to immunofluorescence (IF) using anti-Cb2 (SNO103) (left upper and lower panels) and anti-c-tubulin (middle upper and lower panels) The ima-ges were merged (right upper and lower panels) and showed colocalization of Cb2 and c-tubulin in Cb2 transfected cells.

is concentrated around the Golgi–centrosomal area.

The Cb2 staining demonstrated that Cb2 was

colocal-ized with both RIa and RIIa Based on this, we asked

if Cb2 is associated with both RIa and RIIa by

anti-Cb2(SNO103) immunoprecipitation of T-cell extracts

After washing, the precipitates were extracted with (+)

or without (–) 1 mm 8-CPT-cAMP In this modified

immunoprecipitation procedure the R subunits

associ-ated with the antibody-immobilized Cb2 isoform would

remain in the pellet in the absence of 8-CPT-cAMP

while they would be released to the supernatant in the

presence of 8-CPT-cAMP The precipitates and

supern-atants were analysed by SDS⁄ PAGE and

immunoblot-ting using anti-RIIa and anti-RIa (Fig 3B, upper panel and lower panels) This demonstrated that both RIa and RIIa are immunoprecipitated by anti-Cb2(SNO103) To confirm this interaction, the same lysates were immunoprecipitated with anti-RIa and anti-RIIa The precipitates were extracted with 8-CPT-cAMP as described in Fig 3B The resulting samples were subjected to immunoblotting and incubated with anti-Cmono (Fig 3C) or anti-PKAbcat (Fig 3D) Immunoreactive bands of 40 and 47 kDa which were released by 8-CPT-cAMP were detected in both immu-noprecipitates implying that Ca1 and Cb2 are both asso-ciated with RIa and RIIa in human T cells This

Anti-C β2

Anti-RII α Merge

Merge Anti-RI α

Anti-C β2

A

Anti-Cβ2

IP:

cAMP

-IR IgG

P S P S P

- + +

-50 50

Anti-RIIα IP:

cAMP

-IR IgG

P

- +

50 S

+ + +

RIα

Cα1

Anti-RIα IP:

cAMP + +

IR IgG

P S S P

50 S +

Cα1

Cβ2

D

Fig 3 Cb2 colocalizes and associates with both RIa and RIIa to form PKAI and PKAII in T cells (A) Confocal laser immunofluorescence of human T cells using anti-Cb2(SNO103) in combination with anti-RIa and anti-RIIa Human T cells were incubated with anti-Cb2 and FITC-con-jugated secondary antibody (green, upper and lower left panels), RIa and TRITC secondary antibody (red, upper middle panel) and anti-RIIa and TRITC secondary antibody (red, lower middle panel) Right upper and lower panels show merges of Cb2 and RIa and Cb2 and anti-RIIa, respectively (B) Human T-cell lysates were immunoprecipitated using anti-Cb2(SNO103) The precipitated proteins were washed three times, then extracted using either buffer with (+) or without (–) 8-CPT-cAMP Irrelevant rabbit IgG was used as a control (IR IgG) The result-ing pellets (P) and supernatants (S) were subjected to SDS ⁄ PAGE and immunoblotted using anti-RIIa (upper) or anti-RIa (lower) (C) Human T-cell lysates were immunoprecipitated using anti-RIIa, washed and subsequently extracted using either buffer (–) or buffer containing 8-CPT-cAMP (+) Irrelevant rabbit IgG was used as a control (IR IgG) The resulting pellets (P) and supernatants (S) were subjected to SDS ⁄ PAGE and immunoblotted using monoclonal anti-C (D) Human T-cell extracts were immunoprecipitated using anti-RIa The precipitate was extracted first with buffer, then with buffer containing 8-CPT-cAMP Irrelevant mouse IgG (IR IgG) was used as a control The resulting samples were subjected to SDS ⁄ PAGE and immunoblotted using anti-PKAbcat.

observation prompted us to ask whether Ca1 and Cb2

may be associated on the same holoenzyme

Cb2 and Ca1 may form part of the same

holoenzyme

Lysates of human T-cells were immunoprecipitated

using anti-Cb2(SNO103), and the resulting precipitates

were extracted with (+) or without (–) 8-CPT-cAMP

and the resulting samples analysed by SDS⁄ PAGE and

immunoblotting using anti-Cmono In the absence of

8-CPT-cAMP anti-Cb2(SNO103) precipitated both a

47- and a 40 kDa C When the precipitate was

extrac-ted with 8-CPT-cAMP, the 47 kDa Cb2 remained in

the precipitate while the 40 kDa Ca1 was released into

the extract (Fig 4A) This demonstrated that Cb2 was

directly immunoprecipitated using anti-Cb2(SNO103),

while immunoprecipitation of Ca1 was dependent

on an intact R–C interaction, thereby demonstrating

association of Cb2 to Ca1 through an R subunit This

is consistent with what was demonstrated in Fig 2B,

that anti-Cb2(SNO103) specifically immunoprecipitates

free Cb2 and not free Ca1 The most likely

explan-ation for these results is the presence of holoenzymes

containing both Ca1 and Cb2 However, the

coimmu-noprecipitation could also be the result of pull-down

of larger cell structures, protein clusters or A-kinase

anchoring proteins that have the ability to associate with more than one PKA holoenzyme at the time If

so, anti-Cb2-dependent pull-downs of holoenzymes both made up of R subunits associated with two Cb2 or two Ca1 subunits may occur We find this explanation less likely, as coimmunoprecipitation was observed also in the presence of RIPA-buffer (contain-ing 0.1% SDS) In addition, pull-down was also dem-onstrated in the presence of Ht-31, a peptide known

to disrupt the R to AKAP interaction (results not shown) Finally, the precipitates were analysed for PKA-specific kinase activity, as shown in Fig 4B This demonstrated that precipitates extracted with 8-CPT-cAMP contained 8-CPT-cAMP-inducible PKA activity that could be inhibited by protein kinase inhibitor (PKI)

In the presence of cAMP approximately one-third of the PKI-inhibited activity was released into the extract, indi-cating that the precipitated Cb2 colocalized with Ca1 on RIa and RIIa in a ratio of 2 : 1 Of the total PKA kin-ase activity,  5% could be immunoprecipitated using anti-Cb2(SNO103) (data not shown) However, this im-munoprecipitation was not complete, as flow-through still contained Cb2 as judged by immunoblot analysis and only 20–30% of Cb2 activity in Cb2-transfected 293T cells was precipitated Taken together it is there-fore reasonable to assume that Cb2 activity may consti-tute more than 20% of total PKA activity in T-cells

It has been demonstrated that Ca and Cb when combined with RIIa will form PKA holoenzymes (RIIa2Ca12 and RIIa2Cb12) with different relative association constants (Ka) for cAMP [12] This implies that C subunit isoforms influence R subunit cAMP-binding features which may have biological implica-tions for cAMP effects conveyed by PKA in vivo Furthermore, it has been shown that cAMP is formed and degraded during perturbation of the T-cell antigen receptor complex in conjunction with the anti-CD28 marker [7,11,13,14] During this process, PKAI is translocated to the antigen receptor complex and acti-vated to phosphorylate several substrates important for regulating antigen-dependent activation of T cells

to proliferation and clonal expansion These substrates include PLC-c1⁄ 2 [15], p50csk [16] and Raf-1 [17] in the early phase of the stimulatory process and regula-tion of interleukin-2 producregula-tion through phosphoryla-tion of the nuclear factor of activated T cells [18] and nuclear factor jB [19] The fact that human T cells express two distinct C subunits (Ca1 and Cb2) which may be associate on the same R subunits RIa and RIIa suggest the existence of novel PKA holoenzymes with unique properties in T cells Such properties may

be important for PKA holoenzyme features such as localization and substrate preferences

Anti-Cβ2 IP:

IR IgG

cAMP 943 ±96 50.9 ±11 817 ±214 313 ±87.7 50.7 ±35.9 7.5 ±5.0

PKI 35.7 ±2.5 nd 12.7 ±4.09 9.1 ±7.7 nd nd

Kinase activity (pmol ATP/mL/min)

Cα1

Cβ2 50

37

B

A

Fig 4 Colocalization of Cb2 and Ca1 on the same PKA

holoen-zyme (A) Human T cells were lysed and immunoprecipitated using

anti-Cb2(SNO103) The precipitates were extracted with (+) or

with-out (–) 8-CPT-cAMP and the pellet (P) and supernatants (S)

analysed for immunoreactive C subunits by SDS ⁄ PAGE and

immu-noblotting using monoclonal anti-C Immunoprecipitation using

irre-levant rabbit IgG (IR IgG) was used as a control (B) Analysis of

kinase activity in immunoprecipitates from (A) using a PKA-specific

assay Cyclic AMP (5 lm) was added to samples not extracted with

8-CPT-cAMP, and PKI was included to detect background kinase

activity.

Experimental procedures

Antibodies

In total, six different anti-C Igs were used (Table 1) These

comprised three different rabbit polyclonal commercial

anti-C Igs with limited isoform specificity (anti-PKAacat,

anti-PKAbcat and anti-PKAccat; catalogue numbers sc903,

sc904, sc905; Santa Cruz Biotechnology, Santa Cruz, CA,

USA); a monoclonal anti-C antibody (anti-Cmono;

cata-logue number 610980, BD Biosciences); a rabbit was

immunized using two Cb2-specific peptides, NH2-MAY

REPPCNQYTGTTTALQ-CONH2 and NH2-CFHRHSKG

TAHDQKTALEND-CONH2 generating a

splice-variant-specific antibody designated anti-Cb2(SNO103; a

Cb-speci-fic antibody (expected to recognize all Cb splice variants

but not to cross-react with Ca) was generated by

immun-izing a rabbit using the synthetic peptide NH2-QNNA

GLEDFERK-CONH2and designated as anti-Cb(SNO157)

Peptide synthesis and rabbit immunizations were performed

by Eurogentec SA (Seraing, Belgium) IgG was purified

from anti-Cb2(SNO103) anti-Cb(SNO157) antibodies using

protein A sepharose (Amersham Biosciences, Oslo,

Nor-way, catalogue number 17-0780-01) as described by the

manufacturer A previously described mouse monoclonal

anti-RIa antibody [7] was used for immunoprecipitation

and immunoblotting of RIa For detection of RIIa by

im-munoblotting, a mouse mAb (anti-RIIa; catalogue number

612243; BD Biosciences, Erembodegem, Belgium) was used,

and for immunoprecipitation of RIIa a rabbit anti-peptide

Ig previously described was used [20] Anti-c-tubulin,

mouse monoclonal IgG (Sigma Aldrich, Oslo, Norway,

catalogue number T6557) was used for labelling

centro-somes

Purification of T cells

Human T cells were purified from healthy blood donors

using negative selection Written approval was obtained

from all donors of blood for use in research Briefly,

mono-nuclear cells were isolated from human blood (Ullevaal

University Hospital Blood Centre, Oslo, Norway) using

density gradient centrifugation (Lymphoprep; Nycomed,

Oslo, Norway) The cells were enriched for T cells by

neg-ative selection using anti-CD14 and anti-CD19 Ig-coated

magnetic beads (Dynal, Oslo, Norway) Routinely,

anti-CD3 labelling demonstrated > 90% anti-CD3 positive cells by

flow cytometry

Construction of expression vectors and culture

and transfection of 293T cells

Human cDNAs encoding full-length Ca1 and Cb2 were

amplified from NT-2 cell mRNA using the Promega

Reverse Transcription system and the Pfu-ultra

amplifica-tion system (Stratagene, La Jolla, CA, USA) as described

by the manufacturers The amplified products were cloned into the mammalian expression vector pEF-DEST51 (Invi-trogen, catalogue number 12285-011), and verified to encode full-length native Ca1 and Cb2 by sequencing (Medigenomix Gmbh, Martinsried, Germany) The human kidney 293T cells were grown in RPMI-1640 medium sup-plemented with 5% fetal calf serum Semi-confluent cells were transfected using LipofectAMINE2000 (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer

Lysis of cells, immunoprecipitation, immunoblotting and PKA enzymatic assay Cells were lysed either in RIPA buffer [10 mm Tris⁄ HCl

pH 7.5, 1 mm EDTA, 1% (v⁄ v) Triton X-100, 0.1% (w ⁄ v) SDS, 0.1% (w⁄ v) Na-deoxycholate and 100 mm NaCl] con-taining 1 mm dithiothreitol, 1 mm phenlymethylsulfonyl fluoride and protease inhibitor cocktail (Roche Diagnostics, Oslo, Norway) for nonenzymatic assays, or in 1% Triton buffer [25 mm Mes, pH 6.5, 100 mm NaCl, 5 mm EDTA, 1.0% (v⁄ v) Triton X-100 with 1 mm sodium orthovanadate,

1 mm phenlymethylsulfonyl fluoride, 10 mm sodium pyro-phosphate, and 50 mm sodium fluoride] for enzymatic

assays Lysates were cleared by centrifugation at 15000 g,

30 min, 4
C, and subsequently incubated with primary antibody [anti-Cb2(SNO103) 320 lgÆmL)1, mouse anti-RIa 2.5 lgÆmL)1, rabbit anti-RIIa serum diluted 1 : 100], for

2 h to overnight Antibody–antigen complexes were precipi-tated using either Dynabeads protein G (Dynal, catalogue number 100.04), anti-mouse agarose beads or anti-rabbit agarose beads (Sigma, catalogue number A6531, A1027), washed three times using appropriate buffer and extracted with buffer in the presence or absence of 1 mm 8-CPT-cAMP For immunoblotting, proteins were separated by SDS⁄ PAGE and transferred to poly(vinylidene difluoride) (PVDF) membranes by electroblotting Membranes were blocked in 5% (w⁄ v) skimmed milk powder in Tris-buffered saline containing 0.1% (v⁄ v) Tween-20 (TBST) for 1 h at room temperature, and then incubated for 1 h at room temperature or overnight at 4
C with the appropriate pri-mary antibodies diluted in TBST Membranes were washed for about 1 h in TBST and further incubated with horse-radish peroxidase-conjugated secondary antibodies (MP Biomedicals, Irvine, CA, USA, catalogue number 55689, 55563) Membranes were washed and finally developed using SuperSignal West Pico Chemiluminescent (Pierce Biotechnology, Rockford, IL, USA) cAMP-dependent pro-tein kinase activity was determined as described previously [21], using either untreated lysates or immunoprecipitates

Indirect immunofluorescence Resting human T cells were allowed to attach to poly(l-lysine) coated cover slips for 30 min at room temperature,

followed by fixation in 3% (v⁄ v) paraformaldehyde Cells

were permeabilized using 0.1% (v⁄ v) Triton X-100 in

NaCl⁄ Pi(PBST), followed by blocking with 2% (w⁄ v) BSA

in PBST Cells were incubated with primary antibody in

PBST⁄ BSA for 30 min, washed three times in PBST before

incubation with fluorochrome-conjugated secondary

anti-bodies for 30 min; FITC-conjugated goat antirabbit or

Tetramethylrhodamin-isothiocyanate-conjugated goat

anti-mouse (Sigma, catalogue number F0382, T5393) diluted

1 : 500 in PBST⁄ BSA Finally, cells were washed four time

for 5 min in PBST–BSA and the samples were mounted using

the Dako fluorescent mounting medium (Dakocytomation,

Oslo, Norway; catalogue number S3023) Cells were

exam-ined with a Nikon Labophot microscope (Nikon Instruments

Europe, Badhoevedorp, Netherlands) equipped with an

epifluorescence attachment and a Bio-Rad (Bio-Rad

Labora-tories Ltd, Hemel Hempstead, UK) MRC 600 confocal laser

scan unit with a krypton⁄ argon laser, a K1 double dichroic

excitation filter block, and a K2 dichroic emission filter block

(Bio-Rad) Transfected 293T cells were analysed in a similar

manner, however, they were fixed using 100% (v⁄ v)

meth-anol and visualized using an Olympus (Olympus Norge A/S,

Oslo, Norway) BX61 microscope attached to a digital

camera

Acknowledgements

We appreciate the technical assistance of S Eikvar

This work was supported by grants from the

Nor-wegian Cancer Society, the NorNor-wegian Research

Coun-cil, Novo Nordisk Foundation, the Anders Jahre

Foundation, the Throne Holst Foundation and the

Letten Saugstad Foundation

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