H11

Kinase Classification: Atypical Group: Likely Non-Kinases

H11: A cautionary tale in ascribing protein kinase activity to novel proteins

The human H11 (HSPB8, Hsp22) is a member of the 'small heat-shock protein' family of chaperones (HSP20). It was reported to have protein kinase activity, with evidence that at first seemed compelling, but that has unraveled with time. It serves as a cautionary tale for other proteins that have biochemically-reported kinase activity but lack homology to other kinases.

Evidence of kinase activity came from both the human H11 protein, and a viral homolog, ICP10:

ICP10: the case for kinase activity

In Herpes Simplex Virus 2 (HSV-2), the ribonucleotide reductase protein, ICP10, has an N-terminal extension containing a small heat-shock protein (HSP20) domain. The equivalent HSV-1 protein (ICP6) lacks this extension. The full-length ICP10 and the N-terminal fragment were able to autophosphoryate and to transphosphorylate calmodulin and histone [1, 2], but ICP6 had no activity. Protein produced in human cells, purified by SDS-PAGE and renatured could still autophosphorylate, and the protein bound the ATP analog, FSBA. A manual protein alignment showed partial conservation of ePK-conserved catalytic motifs [2]

H11: the case for kinase activity

Following on the ICP10 work, H11-GST fusion protein was purified from bacteria and shown to autophosphorylate in an in vitro kinase assay [3]. Similarly, expression of H11 in human 293 cells followed by immunoprecipitation with an anti-H11 antibody revealed a 25kDa phosphoprotein (at the H11 size), which was not observed when the predicted catalytic lysine was mutated. This phosphorylation was dependent on Mn2+, and to a lesser extent, Mg2+, similar to the case for ICP10. Transphosphorylation of MBP by H11 immunopreciptates was also seen in a mouse overexpressing H11 [4].

ICP10: the case against

The sequence alignment and identification of catalytic motifs is now clearly an artifact of oversearching for patterns, by observation of the original alignment (Fig 3 from [2]). Since the protein is now known to form a HSP20 fold rather than an PKL fold, the catalytic motifs are further invalidated. Another alignment purporting to show homology to the atypical kinase FASTK [3] looks equally dubious, and is not supported by a comparison that includes many FASTK homologs (i.e. the aligning regions are not well conserved).

Deletion studies show that deletion or mutation of several predicted catalytic motifs did not alter the observed kinase activity [5]

The kinase activity of the bacterially-purified protein was split into two activities, autophosphorylation and transphosphorylation. The transphosphorylation of histones and other proteins mapped in deletion studies to the ribonuclease portion of the protein rather than the HSP20 N-terminus, and could even be separated from the ribonucleotide reducase fragment by ion-exchange chromatography, indicating that a Mn2+-sensitive contaminating E. coli kinase co-purified with the ICP10 [6]. While E. coli does not have any ePK kinases, this casts some doubt on the use of bacterial overexpression systems to claim kinase activity in novel proteins.

Cooper et al [7] mapped the autophosphorylation activity to the N-terminal 292 AA of the protein, and showed that the FSBA binding was outside of this region, negating that as evidence for kinase activity.

A further detailed analysis by Langelier et al [8] showed that highly-purified bacterially-produced protein had an extremely low rate of phosphorylation, and that protein purified from eukaryotic cells was phosphorylated by the abundant and ubiquitous eukaryotic kinase, CK2. In a detailed series of experiments, it was shown that CK2 could co-purify with ICP10 and that it could apparently migrate on the blot to re-associate with the ICP10 substrate.

H11: the case against

As with ICP10, further purification of the H11 protein dramatically reduced the observed autophosphorylation activity (to requiring a 12 hour incubation in rat [9] or showing only 50 ppm of phosphoprotein in human [10], and transphosphorylation was also ablated [10]. In addition, the putative catalytic lysine is not associated with ICP10 activity, and is not a validated catalytic residue, so it is as likely that its association with kinase activity is due to a role in binding an associated protein kinase, such as CK2.

References

1. Luo JH, Smith CC, Kulka M, and Aurelian L. A truncated protein kinase domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) expressed in Escherichia coli. J Biol Chem. 1991 Nov 5;266(31):20976-83.
2. Chung TD, Wymer JP, Smith CC, Kulka M, and Aurelian L. Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10). J Virol. 1989 Aug;63(8):3389-98. DOI:10.1128/JVI.63.8.3389-3398.1989 |
3. Smith CC, Yu YX, Kulka M, and Aurelian L. A novel human gene similar to the protein kinase (PK) coding domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) codes for a serine-threonine PK and is expressed in melanoma cells. J Biol Chem. 2000 Aug 18;275(33):25690-9. DOI:10.1074/jbc.M002140200 |
4. Depre C, Hase M, Gaussin V, Zajac A, Wang L, Hittinger L, Ghaleh B, Yu X, Kudej RK, Wagner T, Sadoshima J, and Vatner SF. H11 kinase is a novel mediator of myocardial hypertrophy in vivo. Circ Res. 2002 Nov 29;91(11):1007-14. DOI:10.1161/01.res.0000044380.54893.4b |
5. Luo JH and Aurelian L. The transmembrane helical segment but not the invariant lysine is required for the kinase activity of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10). J Biol Chem. 1992 May 15;267(14):9645-53.
6. Conner J, Cooper J, Furlong J, and Clements JB. An autophosphorylating but not transphosphorylating activity is associated with the unique N terminus of the herpes simplex virus type 1 ribonucleotide reductase large subunit. J Virol. 1992 Dec;66(12):7511-6. DOI:10.1128/JVI.66.12.7511-7516.1992 |
7. Cooper J, Conner J, and Clements JB. Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase. J Virol. 1995 Aug;69(8):4979-85. DOI:10.1128/JVI.69.8.4979-4985.1995 |
8. Langelier Y, Champoux L, Hamel M, Guilbault C, Lamarche N, Gaudreau P, and Massie B. The R1 subunit of herpes simplex virus ribonucleotide reductase is a good substrate for host cell protein kinases but is not itself a protein kinase. J Biol Chem. 1998 Jan 16;273(3):1435-43. DOI:10.1074/jbc.273.3.1435 |
9. Chowdary TK, Raman B, Ramakrishna T, and Rao CM. Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperone-like activity. Biochem J. 2004 Jul 15;381(Pt 2):379-87. DOI:10.1042/BJ20031958 |
10. Kim MV, Seit-Nebi AS, Marston SB, and Gusev NB. Some properties of human small heat shock protein Hsp22 (H11 or HspB8). Biochem Biophys Res Commun. 2004 Mar 19;315(4):796-801. DOI:10.1016/j.bbrc.2004.01.130 |

--Gerard 15:12, 15 April 2010 (PDT)