- Email: [email protected]
Cloning and preliminary characterization of a 105 kDa protein with an N-terminal kinase-like domain
Biochimica et Biophysica Acta 1517 (2000) 148^152
www.elsevier.com/locate/bba
Short sequence-paper
Cloning and preliminary characterization of a 105 kDa protein with an N-terminal kinase-like domain Simon C.H. Liu a , William S. Lane b , Gustav E. Lienhard b
a;
*
a Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA Harvard Microchemistry Facility, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
Received 11 July 2000 ; received in revised form 7 September 2000; accepted 7 September 2000
Abstract In the course of searching for proteins that interact with protein kinase B in 3T3-L1 adipocytes, we isolated a 105 kDa protein from 3T3L1 adipocytes. Peptides sequenced from the protein were found to be present in several expressed sequence tags. A cDNA containing one of these expressed sequence tags was sequenced and appears to contain the entire coding region. Computer analysis revealed a potential protein kinase domain at the N-terminus; however, the first subdomain and several invariant residues characteristic of protein kinases are absent. An antibody was raised against a peptide from the 105 kDa protein. By immunoblotting, it was found that the protein was widely expressed in mouse tissues, and concentrated in the cytosol and low density microsome fractions of 3T3-L1 adipocytes. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Protein kinase ; Adipocyte
We have been searching for proteins that interact with protein kinase B (PKB, also known as Akt) in 3T3-L1 adipocytes, by the approach of coimmunoprecipitation and aa sequence analysis. During this investigation, we have identi¢ed and partially characterized a novel 105 kDa protein (designated p105). Although we later determined that p105 does not associate with PKB, the presence of a kinase-like domain in p105 suggests it is a novel signaling protein. 3T3-L1 adipocytes, cultured as described in [1], were solubilized in 1 ml of the following lysis bu¡er per 10 cm plate: 20 mM HEPES, 120 mM KCl, 10 mM L-glycerolphosphate, 5 mM MgCl2 , 3% (w/v) Thesit, 100 nM okadaic acid, and 1/100 dilution of protease inhibitor cocktail (Sigma P8340), pH 7.4. The lysate was cleared by centrifugation at 20 000Ug for 10 min. Cleared lysate from 10 plates (approximately 70 mg protein) was immunoadsorbed with 100 Wg PKB antibody (Santa Cruz, H136, against aa 345^480 of human PKBK, reacts with both PKBK and L), and the immunoadsorbate was collected on Abbreviations : aa, amino acid; est, expressed sequence tag; PKB, protein kinase B; RACE, rapid ampli¢cation of cDNA ends; SDS^PAGE, sodium dodecyl^polyacrylamide gel electrophoresis * Corresponding author. Fax: +1-603-650-1128; E-mail : [email protected]
50 Wl protein A-Sepharose (Pharmacia). The protein ASepharose was washed ¢ve times with lysis bu¡er, and the bound proteins were released at 100³C for 5 min in SDS^PAGE sample bu¡er containing 20 mM DTT and 1/100 dilution of protease inhibitor cocktail. The proteins were separated on a 5^15% gradient gel and stained with Colloidal Coomassie Blue (Novex). A 105 kDa protein was detected. It was not present in control immunoprecipitations where either anti-PKB was replaced with irrelevant rabbit IgG, or cell lysate was replaced with lysis bu¡er. Peptides from the 105 kDa protein were generated by in gel tryptic digestion and sequenced by microcapillary reverse-phase high pressure liquid chromatography directly coupled to a Finnigan LCQ quadrupole ion trap mass spectrometer, as in [2]. Twelve peptides were identi¢ed ; each was present in one or more of the following four mouse ests and one human est: AA655289, AI508998, AI526559, AA451130, and R51708, respectively. AA655289 was purchased from Research Genetics and sequenced in both directions; all 12 peptides were present in the translated sequence (Fig. 1). AI508998 and AI526559 were obtained from Genome Systems and also sequenced; both are identical to the 3P end of AA655289 (data not shown). AA451130 was also purchased from Genome Systems, but did not contain the expected est. A primer derived from the 5P end of
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 2 3 4 - 7
BBAEXP 91482 6-12-00
S.C.H. Liu et al. / Biochimica et Biophysica Acta 1517 (2000) 148^152
149
Fig. 1. cDNA and amino acid sequence of p105. The cDNA sequence of p105 was determined by sequencing mouse est AA655289 in both directions using the Applied Biosystems 373 DNA Sequencing System. Additional sequence at the 5P end (lower case letters) was obtained by 5P RACE. Underneath the cDNA sequence is the predicted aa sequence of p105. The tryptic peptides sequenced by mass spectrometry are highlighted in bold and are located at the following aa sites : 73^87, 114^126, 201^207, 263^271, 293^299, 411^418, 419^430, 467^474, 478^484, 537^552, 717^731 and 732^741.
AA655289 was used in 5P RACE (System Version 2.0, Gibco BRL) of 3T3-L1 adipocyte mRNA, which had been isolated with the FastTrak kit from Invitrogen. This procedure yielded an additional 26 nucleotides of highly GC rich sequence from the 5P end of AA655289 (Fig. 1, ¢rst 26 nucleotides). The sequence of p105
cDNA has been submitted to GenBank under accession number AF276514. Translation of the cDNA sequence yields an open reading frame of 806 aa starting from the ¢rst AUG codon (Fig. 1). It is uncertain as to whether this Met is the initiation Met for translation. The 5P upstream sequence con-
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150
S.C.H. Liu et al. / Biochimica et Biophysica Acta 1517 (2000) 148^152
Fig. 2. Alignment of the kinase domains of C-TAK1 kinase (upper sequence) and p105 (lower sequence). The aa sequence of p105 was compared with the non-redundant database by a BLAST search. The kinase domain of C-TAK1 kinase was one of a number of kinases that showed moderate similarity with a region of p105. The aa sequences designated by upper case letters in the ¢gure depict the region of similarity as de¢ned by the BLAST search. In this region the identity and similarity between C-TAK1 kinase and p105 are 26 and 42%, respectively. Identical and similar residues are highlighted in black and gray, respectively. The kinase subdomains I^XI and the consensus motifs that de¢ne these, as described by Hanks and Hunter [6], are above the sequences. For the consensus motifs the designations are: capital letter, invariant aa; small letter, almost invariant aa; o, non-polar aa; *, polar aa; +, small aa.
tains no in-frame stop codon. The sequence context of the putative initiation AUG is a relatively common initiation sequence context for mouse, although not the most frequent one [3]. A BLAST search of the non-redundant database with the p105 protein sequence identi¢ed three predicted proteins with considerable similarity to p105 over almost their full length. These are: in Drosophila, AAF56933, 873 aa, 48% identity; in Caenorhabditis elegans, CAB01444, 820 aa, 36% identity ; and in Arabidopsis, AAD32806, 604 aa, 35% identity. The ¢rst two predicted proteins have an N-terminal sequence similar to the Nterminal sequence of p105 and are also approximately the same size as p105. These facts suggest that the assumed initiation Met for p105 is the correct one. In addition, the existence of similar proteins in insect, worm, and plant suggests a fundamental role for p105. The calculated
size of p105 is 89104 Da, somewhat less than the 105 kDa size estimated from SDS^PAGE. The aa sequence of p105 was analyzed for domains with the program SMART [4]; a kinase domain was predicted at aa 31^205. In addition, the BLAST search of p105 against the non-redundant database revealed moderate similarity between this region of p105 and the kinase domains of a number of established protein kinases. Fig. 2 shows the alignment between this region of p105 and the kinase domain of one such kinase, mouse C-TAK1 protein kinase (AAF64455) [5]. Virtually all kinase domains are characterized by 12 subdomains (designated by Roman numerals I^XI, including VIA and VIB), which are regions not interrupted by large insertions and consisting of a pattern of conserved aa residues [6]. As shown in Fig. 2, the kinase domain of C-TAK1 possesses all these subdo-
BBAEXP 91482 6-12-00
S.C.H. Liu et al. / Biochimica et Biophysica Acta 1517 (2000) 148^152
mains. On the other hand, the putative kinase domain of p105 lacks subdomain I. Moreover, the putative kinase domain of p105 is missing certain residues that are invariant or almost invariant in other subdomains, speci¢cally, Lys in subdomain II, Glu in subdomain III, Asp and Asn in subdomain VIB, and Gly in subdomain VII (Fig. 2). The absence of subdomain I and these invariant residues strongly suggest that p105 is not a functional protein kinase. The proteins similar to p105 in Drosophila, C. elegans, and Arabidopsis described above also have a kinaselike domain that lacks subdomain I and the other invariant residues missing from p105. The Lys of subdomain II is generally considered essential for kinase activity [6]. However, a kinase has recently been described in which this Lys is missing, and the essential Lys is provided by a Lys in subdomain I [7]. Thus, rigorous determination of whether p105 has kinase activity will require expression of the recombinant protein and testing of it for activity against a variety of substrates. In order to immunoblot and immunoprecipitate p105, we generated a rabbit antiserum against a peptide corresponding to the C-terminal 18 aa of p105, with a Cys at the amino-terminus for conjugation to the carrier, and a¤nity puri¢ed the antibodies on immobilized peptide, as described in [8]. Immunoblotting of a 3T3-L1 adipocyte lysate with the antibody revealed a single strong band at 105 kDa (data not shown). We performed a number of experiments in order to determine whether p105 was associated with PKB in 3T3-L1 adipocytes. The results of these showed that p105 does not associate with PKB and that, most likely, our initial isolation of p105 in the immunoprecipitate of PKB was due to direct binding of p105 to the Santa Cruz H-136 PKB antibody. Since this outcome is a negative one, the results will only be brie£y described. First, the Santa Cruz H-136 antibody immunoprecipitated only approximately 1% of the p105 in the 3T3-L1 lysate. Second, the Santa Cruz H-136 antibody immunoprecipitated as much p105 from a lysate largely depleted of PKB by prior immunoprecipitation as it did from undepleted lysate.
Fig. 3. Tissue distribution of p105. SDS samples (150 Wg protein) of mouse tissues were separated by SDS^PAGE and immunoblotted as described in [12], with the p105 antibody at 1 Wg per ml. BAT, brown adipose tissue; WAT, white adipose tissue. A repetition of this experiment gave similar results.
151
Fig. 4. Subcellular distribution of p105. SDS samples of subcellular fractions from basal and insulin-treated (1 WM for 15 min) 3T3-L1 adipocytes were immunoblotted for p105. Lanes contained 20 Wg protein except the plasma membrane lanes, which contained 10 Wg. TOT, total lysate; LDM, low density microsomes ; HDM, high density microsomes ; PM, plasma membrane ; M/N, mitochondria/nuclei; CYT, cytosol. A repetition of this experiment gave similar results.
Third, no p105 was present in the immunoprecipitates of PKB obtained with two other antibodies against di¡erent regions of PKB (StressGen KAP-PK004, against aa 88^ 100 of human PKBK, and Upstate Biotechnology 06-606, against aa rat 455^469 of PKBL). Fourth, although our antibody against p105 immunoprecipitated approximately 75% of the p105 in a 3T3-L1 lysate, no PKB was coimmunoprecipitated. To determine the tissue distribution of p105, SDS samples of mouse tissues were prepared as described in [2] and immunoblotted for p105 (Fig. 3). The p105 protein was present in all tissues examined. Two control experiments provided additional evidence that the protein detected by immunoblotting was p105 (data not shown). First, upon immunoblotting the SDS samples of brain, liver, kidney, and spleen with both preimmune serum and immune serum at 1/500 dilution, the p105 band was detected only with the immune serum. Second, when the p105 peptide used for immunization was included at 10 Wg per ml with the immune serum in immunoblotting these samples, the p105 band was not detected. The widespread tissue distribution of p105 suggests that it has a function required by all cells. To determine the subcellular distribution of p105, basal and insulin-treated 3T3-L1 adipocytes were fractionated as described in [9], and the subcellular fractions were immunoblotted for p105 (Fig. 4). The protein was present primarily in the cytosol and in the low density microsomes. The latter consists largely of endosomes, fragmented Golgi, and cytoskeletal elements [10,11]. Insulin treatment did not alter the subcellular distribution of p105. This result indicates that p105 is a soluble protein that can bind to a membrane or cytoskeletal site. In conclusion, p105 is a widely expressed protein with an N-terminal kinase-like domain that probably has no kinase activity. The function of p105, and especially its kinase-like domain, remains to be determined. Since each protein kinase interacts with and phosphorylates a limited number of speci¢c substrate proteins, it is possible that p105 binds to, but does not phosphorylate, a limited number of proteins. If these proteins are normally phosphorylated by another kinase, association with p105 may inhibit their phosphorylation. Alternatively, since many kinases
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are activated by phosphorylation by other kinases, it is possible that p105 competes with a particular kinase for binding to its upstream kinase. In either case, p105 would have a regulatory e¡ect on protein phosphorylation. Just after this manuscript initially was submitted, the amino-terminal sequence (283 aa) of the human homolog of p105 appeared in the database (accession number AAF81422) [13]. This portion of human p105 is 94% identical to the same region of mouse p105. The gene for human p105 has been shown to be located on the chromosome 11q13 region [13]. We are indebted to Kerry A. Pierce for the mass spectrometry and to the late Barbara E. Crute for guidance in DNA sequencing. This research was supported by grant DK42816 from the National Institutes of Health. References [1] S.C. Frost, M.D. Lane, J. Biol. Chem. 260 (1985) 2646^2652.
[2] G. LeRoy, A. Loyala, W.S. Lane, D. Reinberg, J. Biol. Chem. 275 (2000) 14787^14790. [3] M.E. Dalphin, C.M. Brown, P.A. Stockwell, W.P. Tate, Nucleic Acids Res. 26 (1998) 335^337. [4] J. Schultz, F. Milpetz, P. Bork, C.P. Ponting, Proc. Natl. Acad. Sci. USA 95 (1998) 5857^5864. [5] C.Y. Peng, P.R. Graves, S. Ogg, R.S. Thoma, M.J. Byrnes, Cell Growth Di¡er. 9 (1998) 197^208. [6] S.K. Hanks, T. Hunter, FASEB J. 9 (1995) 576^596. [7] Xu Be, J.M. English, J.L. Wilsbacher, S. Stippec, E.J. Goldsmith, M.H. Cobb, J. Biol. Chem. 275 (2000) 16795^16801. [8] L. Lamphere, G.E. Lienhard, Endocrinology 131 (1992) 2196^2202. [9] N.J. Morris, S.A. Ross, W.S. Lane, S.K. Moestrup, C.M. Petersen, S.R. Keller, G.E. Lienhard, J. Biol. Chem. 273 (1998) 3582^3587. [10] I.A. Simpson, D.R. Yver, P.J. Hissin, L.J. Wardzala, E. Karnieli, L.B. Salans, S.W. Cushman, Biochim. Biophys. Acta 763 (1983) 393^407. [11] S.F. Clark, S. Martin, A.J. Carozzi, M.M. Hill, D.E. James, J. Cell Biol. 140 (1998) 1211^1225. [12] S.C. Liu, Q. Wang, G.E. Lienhard, S.R. Keller, J. Biol. Chem. 274 (1999) 18093^18099. [13] M. van Asseldonk, M. Schepens, D. de Bruijn, B. Janassen, G. Merkx, A.G. van Kessel, Genomics 66 (2000) 35^42.
BBAEXP 91482 6-12-00
www.elsevier.com/locate/bba
Short sequence-paper
Cloning and preliminary characterization of a 105 kDa protein with an N-terminal kinase-like domain Simon C.H. Liu a , William S. Lane b , Gustav E. Lienhard b
a;
*
a Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA Harvard Microchemistry Facility, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
Received 11 July 2000 ; received in revised form 7 September 2000; accepted 7 September 2000
Abstract In the course of searching for proteins that interact with protein kinase B in 3T3-L1 adipocytes, we isolated a 105 kDa protein from 3T3L1 adipocytes. Peptides sequenced from the protein were found to be present in several expressed sequence tags. A cDNA containing one of these expressed sequence tags was sequenced and appears to contain the entire coding region. Computer analysis revealed a potential protein kinase domain at the N-terminus; however, the first subdomain and several invariant residues characteristic of protein kinases are absent. An antibody was raised against a peptide from the 105 kDa protein. By immunoblotting, it was found that the protein was widely expressed in mouse tissues, and concentrated in the cytosol and low density microsome fractions of 3T3-L1 adipocytes. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Protein kinase ; Adipocyte
We have been searching for proteins that interact with protein kinase B (PKB, also known as Akt) in 3T3-L1 adipocytes, by the approach of coimmunoprecipitation and aa sequence analysis. During this investigation, we have identi¢ed and partially characterized a novel 105 kDa protein (designated p105). Although we later determined that p105 does not associate with PKB, the presence of a kinase-like domain in p105 suggests it is a novel signaling protein. 3T3-L1 adipocytes, cultured as described in [1], were solubilized in 1 ml of the following lysis bu¡er per 10 cm plate: 20 mM HEPES, 120 mM KCl, 10 mM L-glycerolphosphate, 5 mM MgCl2 , 3% (w/v) Thesit, 100 nM okadaic acid, and 1/100 dilution of protease inhibitor cocktail (Sigma P8340), pH 7.4. The lysate was cleared by centrifugation at 20 000Ug for 10 min. Cleared lysate from 10 plates (approximately 70 mg protein) was immunoadsorbed with 100 Wg PKB antibody (Santa Cruz, H136, against aa 345^480 of human PKBK, reacts with both PKBK and L), and the immunoadsorbate was collected on Abbreviations : aa, amino acid; est, expressed sequence tag; PKB, protein kinase B; RACE, rapid ampli¢cation of cDNA ends; SDS^PAGE, sodium dodecyl^polyacrylamide gel electrophoresis * Corresponding author. Fax: +1-603-650-1128; E-mail : [email protected]
50 Wl protein A-Sepharose (Pharmacia). The protein ASepharose was washed ¢ve times with lysis bu¡er, and the bound proteins were released at 100³C for 5 min in SDS^PAGE sample bu¡er containing 20 mM DTT and 1/100 dilution of protease inhibitor cocktail. The proteins were separated on a 5^15% gradient gel and stained with Colloidal Coomassie Blue (Novex). A 105 kDa protein was detected. It was not present in control immunoprecipitations where either anti-PKB was replaced with irrelevant rabbit IgG, or cell lysate was replaced with lysis bu¡er. Peptides from the 105 kDa protein were generated by in gel tryptic digestion and sequenced by microcapillary reverse-phase high pressure liquid chromatography directly coupled to a Finnigan LCQ quadrupole ion trap mass spectrometer, as in [2]. Twelve peptides were identi¢ed ; each was present in one or more of the following four mouse ests and one human est: AA655289, AI508998, AI526559, AA451130, and R51708, respectively. AA655289 was purchased from Research Genetics and sequenced in both directions; all 12 peptides were present in the translated sequence (Fig. 1). AI508998 and AI526559 were obtained from Genome Systems and also sequenced; both are identical to the 3P end of AA655289 (data not shown). AA451130 was also purchased from Genome Systems, but did not contain the expected est. A primer derived from the 5P end of
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 2 3 4 - 7
BBAEXP 91482 6-12-00
S.C.H. Liu et al. / Biochimica et Biophysica Acta 1517 (2000) 148^152
149
Fig. 1. cDNA and amino acid sequence of p105. The cDNA sequence of p105 was determined by sequencing mouse est AA655289 in both directions using the Applied Biosystems 373 DNA Sequencing System. Additional sequence at the 5P end (lower case letters) was obtained by 5P RACE. Underneath the cDNA sequence is the predicted aa sequence of p105. The tryptic peptides sequenced by mass spectrometry are highlighted in bold and are located at the following aa sites : 73^87, 114^126, 201^207, 263^271, 293^299, 411^418, 419^430, 467^474, 478^484, 537^552, 717^731 and 732^741.
AA655289 was used in 5P RACE (System Version 2.0, Gibco BRL) of 3T3-L1 adipocyte mRNA, which had been isolated with the FastTrak kit from Invitrogen. This procedure yielded an additional 26 nucleotides of highly GC rich sequence from the 5P end of AA655289 (Fig. 1, ¢rst 26 nucleotides). The sequence of p105
cDNA has been submitted to GenBank under accession number AF276514. Translation of the cDNA sequence yields an open reading frame of 806 aa starting from the ¢rst AUG codon (Fig. 1). It is uncertain as to whether this Met is the initiation Met for translation. The 5P upstream sequence con-
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150
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Fig. 2. Alignment of the kinase domains of C-TAK1 kinase (upper sequence) and p105 (lower sequence). The aa sequence of p105 was compared with the non-redundant database by a BLAST search. The kinase domain of C-TAK1 kinase was one of a number of kinases that showed moderate similarity with a region of p105. The aa sequences designated by upper case letters in the ¢gure depict the region of similarity as de¢ned by the BLAST search. In this region the identity and similarity between C-TAK1 kinase and p105 are 26 and 42%, respectively. Identical and similar residues are highlighted in black and gray, respectively. The kinase subdomains I^XI and the consensus motifs that de¢ne these, as described by Hanks and Hunter [6], are above the sequences. For the consensus motifs the designations are: capital letter, invariant aa; small letter, almost invariant aa; o, non-polar aa; *, polar aa; +, small aa.
tains no in-frame stop codon. The sequence context of the putative initiation AUG is a relatively common initiation sequence context for mouse, although not the most frequent one [3]. A BLAST search of the non-redundant database with the p105 protein sequence identi¢ed three predicted proteins with considerable similarity to p105 over almost their full length. These are: in Drosophila, AAF56933, 873 aa, 48% identity; in Caenorhabditis elegans, CAB01444, 820 aa, 36% identity ; and in Arabidopsis, AAD32806, 604 aa, 35% identity. The ¢rst two predicted proteins have an N-terminal sequence similar to the Nterminal sequence of p105 and are also approximately the same size as p105. These facts suggest that the assumed initiation Met for p105 is the correct one. In addition, the existence of similar proteins in insect, worm, and plant suggests a fundamental role for p105. The calculated
size of p105 is 89104 Da, somewhat less than the 105 kDa size estimated from SDS^PAGE. The aa sequence of p105 was analyzed for domains with the program SMART [4]; a kinase domain was predicted at aa 31^205. In addition, the BLAST search of p105 against the non-redundant database revealed moderate similarity between this region of p105 and the kinase domains of a number of established protein kinases. Fig. 2 shows the alignment between this region of p105 and the kinase domain of one such kinase, mouse C-TAK1 protein kinase (AAF64455) [5]. Virtually all kinase domains are characterized by 12 subdomains (designated by Roman numerals I^XI, including VIA and VIB), which are regions not interrupted by large insertions and consisting of a pattern of conserved aa residues [6]. As shown in Fig. 2, the kinase domain of C-TAK1 possesses all these subdo-
BBAEXP 91482 6-12-00
S.C.H. Liu et al. / Biochimica et Biophysica Acta 1517 (2000) 148^152
mains. On the other hand, the putative kinase domain of p105 lacks subdomain I. Moreover, the putative kinase domain of p105 is missing certain residues that are invariant or almost invariant in other subdomains, speci¢cally, Lys in subdomain II, Glu in subdomain III, Asp and Asn in subdomain VIB, and Gly in subdomain VII (Fig. 2). The absence of subdomain I and these invariant residues strongly suggest that p105 is not a functional protein kinase. The proteins similar to p105 in Drosophila, C. elegans, and Arabidopsis described above also have a kinaselike domain that lacks subdomain I and the other invariant residues missing from p105. The Lys of subdomain II is generally considered essential for kinase activity [6]. However, a kinase has recently been described in which this Lys is missing, and the essential Lys is provided by a Lys in subdomain I [7]. Thus, rigorous determination of whether p105 has kinase activity will require expression of the recombinant protein and testing of it for activity against a variety of substrates. In order to immunoblot and immunoprecipitate p105, we generated a rabbit antiserum against a peptide corresponding to the C-terminal 18 aa of p105, with a Cys at the amino-terminus for conjugation to the carrier, and a¤nity puri¢ed the antibodies on immobilized peptide, as described in [8]. Immunoblotting of a 3T3-L1 adipocyte lysate with the antibody revealed a single strong band at 105 kDa (data not shown). We performed a number of experiments in order to determine whether p105 was associated with PKB in 3T3-L1 adipocytes. The results of these showed that p105 does not associate with PKB and that, most likely, our initial isolation of p105 in the immunoprecipitate of PKB was due to direct binding of p105 to the Santa Cruz H-136 PKB antibody. Since this outcome is a negative one, the results will only be brie£y described. First, the Santa Cruz H-136 antibody immunoprecipitated only approximately 1% of the p105 in the 3T3-L1 lysate. Second, the Santa Cruz H-136 antibody immunoprecipitated as much p105 from a lysate largely depleted of PKB by prior immunoprecipitation as it did from undepleted lysate.
Fig. 3. Tissue distribution of p105. SDS samples (150 Wg protein) of mouse tissues were separated by SDS^PAGE and immunoblotted as described in [12], with the p105 antibody at 1 Wg per ml. BAT, brown adipose tissue; WAT, white adipose tissue. A repetition of this experiment gave similar results.
151
Fig. 4. Subcellular distribution of p105. SDS samples of subcellular fractions from basal and insulin-treated (1 WM for 15 min) 3T3-L1 adipocytes were immunoblotted for p105. Lanes contained 20 Wg protein except the plasma membrane lanes, which contained 10 Wg. TOT, total lysate; LDM, low density microsomes ; HDM, high density microsomes ; PM, plasma membrane ; M/N, mitochondria/nuclei; CYT, cytosol. A repetition of this experiment gave similar results.
Third, no p105 was present in the immunoprecipitates of PKB obtained with two other antibodies against di¡erent regions of PKB (StressGen KAP-PK004, against aa 88^ 100 of human PKBK, and Upstate Biotechnology 06-606, against aa rat 455^469 of PKBL). Fourth, although our antibody against p105 immunoprecipitated approximately 75% of the p105 in a 3T3-L1 lysate, no PKB was coimmunoprecipitated. To determine the tissue distribution of p105, SDS samples of mouse tissues were prepared as described in [2] and immunoblotted for p105 (Fig. 3). The p105 protein was present in all tissues examined. Two control experiments provided additional evidence that the protein detected by immunoblotting was p105 (data not shown). First, upon immunoblotting the SDS samples of brain, liver, kidney, and spleen with both preimmune serum and immune serum at 1/500 dilution, the p105 band was detected only with the immune serum. Second, when the p105 peptide used for immunization was included at 10 Wg per ml with the immune serum in immunoblotting these samples, the p105 band was not detected. The widespread tissue distribution of p105 suggests that it has a function required by all cells. To determine the subcellular distribution of p105, basal and insulin-treated 3T3-L1 adipocytes were fractionated as described in [9], and the subcellular fractions were immunoblotted for p105 (Fig. 4). The protein was present primarily in the cytosol and in the low density microsomes. The latter consists largely of endosomes, fragmented Golgi, and cytoskeletal elements [10,11]. Insulin treatment did not alter the subcellular distribution of p105. This result indicates that p105 is a soluble protein that can bind to a membrane or cytoskeletal site. In conclusion, p105 is a widely expressed protein with an N-terminal kinase-like domain that probably has no kinase activity. The function of p105, and especially its kinase-like domain, remains to be determined. Since each protein kinase interacts with and phosphorylates a limited number of speci¢c substrate proteins, it is possible that p105 binds to, but does not phosphorylate, a limited number of proteins. If these proteins are normally phosphorylated by another kinase, association with p105 may inhibit their phosphorylation. Alternatively, since many kinases
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are activated by phosphorylation by other kinases, it is possible that p105 competes with a particular kinase for binding to its upstream kinase. In either case, p105 would have a regulatory e¡ect on protein phosphorylation. Just after this manuscript initially was submitted, the amino-terminal sequence (283 aa) of the human homolog of p105 appeared in the database (accession number AAF81422) [13]. This portion of human p105 is 94% identical to the same region of mouse p105. The gene for human p105 has been shown to be located on the chromosome 11q13 region [13]. We are indebted to Kerry A. Pierce for the mass spectrometry and to the late Barbara E. Crute for guidance in DNA sequencing. This research was supported by grant DK42816 from the National Institutes of Health. References [1] S.C. Frost, M.D. Lane, J. Biol. Chem. 260 (1985) 2646^2652.
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